Methods and compositions for the treatment of cancer or other diseases

ABSTRACT

The present invention relates to methods and compositions for the treatment of diseases, including cancer, infectious diseases and autoimmune diseases. The present invention also relates to methods and compositions for improving immune function. More particularly, the present invention relates to multifunctional molecules that are capable of being delivered to cells of interest for the treatment of diseases and for the improvement in immune function.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a division of U.S. patent application Ser.No. 13/229,146 filed 9 Sep. 2011, now U.S. Pat. No. 8,748,405, which inturn is a continuation-in-part of U.S. patent application Ser. No.12/879,199 filed 10 Sep. 2010, now abandoned, which in turn is acontinuation-in-part of U.S. patent application Ser. No. 11/966,423filed 28 Dec. 2007, now abandoned. The present application is furtherrelated to and claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application Ser. No. 61/466,086 filed on 22 Mar.2011. application Ser. No. 12/879,199 is further related to and claimspriority under 35 U.S.C. §119(e) to U.S. Provisional Patent ApplicationSer. No. 61/241,764 filed on 11 Sep. 2009. application Ser. No.11/966,423 is further related to and claims priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application Ser. No. 60/897,495 filedon 26 Jan. 2007. Each application is incorporated herein by reference.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

The present invention was made in part with Government support underGrant Numbers R01-89693, R01-100878, R01CA115815, R01CA122976,R01CA115674 and P50CA107399 awarded by the National Institutes ofHealth/National Cancer Institute, Bethesda, Md. The Government hascertain rights in this invention.

SEQUENCE SUBMISSION

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is entitled1954570SequenceListing.txt, created on 2 Jun. 2014, and is 7 kb in size.The information in the electronic format of the Sequence Listing is partof the present application and is incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

The present invention relates to methods and compositions for thetreatment of diseases, including cancer, infectious diseases andautoimmune diseases. The present invention also relates to methods andcompositions for improving immune function. More particularly, thepresent invention relates to multifunctional molecules that are capableof being delivered to cells of interest for the treatment of diseasesand for the improvement in immune function.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedbibliography.

Signal Transducer and Activator of Transcription 3 (Stat3) isconstitutively activated at high frequency (50 to 100%) in diversecancers (Yu and Jove, 2004; Yu et al., 2007; Kortylewski et al., 2005a).Blocking Stat3 in tumor cells induces tumor cell apoptosis, inhibitstumor angiogenesis and abrogates metastasis (Yu and Jove, 2004; Yu etal., 2007; Xie et al., 2004; Xie et al., 2006), and activates antitumorimmune responses (Wang et al., 2004; Kortylewski et al., 2005b). Ourrecent studies further demonstrate that Stat3 is constitutivelyactivated in tumor-stromal myeloid cells, including Gr1⁺ immaturemyeloid cells, DCs, macrophages, NK cell, neutrophils. Activated Stat3inhibits expression of Th-1 type immune responses while promoting tumoraccumulation of T regulatory cells and Th17 cells, compromisingantitumor effects of immune effector cells, such as NK cells,neutrophils and CD8⁺ T cells (Kortylewski et al., 2005b). Blocking Stat3in the immune subsets leads to activation of antitumor immunity andimmune-mediated tumor growth inhibition and tumor regression(Kortylewski et al., 2005b). Our preliminary data further demonstratethat Stat3 is constitutively activated in CD4⁺CD25⁺/Foxp3⁺ T regulatorycells within the tumor stroma. A requirement of Stat3 for expression ofFoxp3, TGFβ and IL-10—the hallmarks of T regulatory cells—in CD4⁺ Tcells has been demonstrated in both animal models and human T cellsobtained from clinical trials (Yu et al., 2007). A recent studyinvolving human melanoma cells has also confirmed a critical role ofStat3 in mediating tumor immune evasion/suppression (Sumimoto et al.,2006).

Stat3 is a point of convergence for numerous tyrosine kinase signalingpathways, which are the most frequently overactive oncogenic pathways intumor cells of diverse origins (Yu and Jove, 2004). The reason Stat3 isalso constitutively-activated in tumor stromal cells is because many ofthe Stat3 target genes encode secreted molecules whose cognate receptorssignal through Stat3 (Yu et al., 2007). For example, Stat3-regulatedproducts such as IL-10, IL-6 and VEGF have their receptors in diversemyeloid cells and T lymphocytes. VEGF and bFGF, both of which alsorequire Stat3 for their expression, activates Stat3 in endothelialcells. Activated Stat3 promotes expression of a wide range of genescritical for tumor cell survival, proliferation, angiogenesis/metastasisand immune suppression. Activated Stat3 also inhibits expressionmultiple genes that are pro-apoptotic, anti-angiogenic and Th-1 typeimmunostimulatory, whose upregulation are critical for anti-cancertherapy (Yu and Jove, 2004; Yu et al., 2007; Kortylewski et al., 2005b).

RNA interference provides compelling opportunities to control geneexpression in cells and siRNAs therefore represent a family of new drugswith broad potential for the treatment of diverse human diseases.Several recent studies have demonstrated the feasibility of in vivosiRNA delivery, leading to therapeutic effects in mouse models (Song etal., 2005; Hu-Lieskovan et al., 2005; McNamara et al., 2006; Kumar etal., 2007; Poeck et al., 2008) and also in non-human-primates (Li etal., 2005; Zimmermann et al., 2006). Nevertheless, efficient in vivotargeted delivery of siRNA into specific cell types, especially those ofimmune origin, which are important constituents of the tumormicroenvironment and active players in promoting tumor progression,remains to be fully explored before the full potential of therapeuticRNA interference can be realized. One promising approach for targeteddelivery of siRNA is the use of aptamers, which areoligonucleotide-based ligands that bind to specific receptors, such asthose on tumor cells (McNamara et al., 2006). Recent studies furtherindicated the ability of specific aptamers to bind and modulate thefunctions of their cognate targets in T cells, leading to potentantitumor immune responses (McNamara et al., 2008). However, whetherthese aptamers can mediate siRNA delivery into T cells remains to bedetermined.

The immune system can serve as extrinsic tumor suppressor (Bui andSchreiber, 2007; Koebel et al., 2007; Shankaran et al., 2001). However,the microenvironment of established tumors is typically characterized bya paucity of tumor-specific CD8⁺ T cells together with an excess ofsuppressive regulatory T cells and myeloid-derived suppressor cells(MDSC) that promote tumor immune evasion (Kortylewski et al., 2005b; Yuet al., 2005; Curiel et al., 2004; Ghiringhelli et al., 2005; Melani etal., 2003). Myeloid cells and other immune cells in the tumormicroenvironment also produce growth factors and angiogenic/metastaticfactors critical for tumor progression (Kujawski et al., 2008). As notedabove, Stat3 is an important oncogenic molecule. The orchestration ofthese processes in the tumor microenvironment is highly dependent on theoncogenic transcription factor, Stat3 (Yu et al., 1995; Bromberg et al.,1999; Yu and Jove, 2004; Darnell, 2002; Yu et al., 2007). In particular,we and others have recently demonstrated a critical role of Stat3 inmediating tumor immune evasion (Wang et al., 2004; Kortylewski et al.2005b; Yu et al., 2007). Activated Stat3 in myeloid cells inhibitsexpression of a large number of immunostimulatory molecules related toTh1-type responses, while promoting production of several keyimmunosuppressive factors (Yu et al., 2007, Kortylewski and Yu, 2008;Kortylewski et al., 2009a) as well as angiogenic factors (Kujawski etal., 2008). In addition, by mediating signaling of certain cytokines andgrowth factors, notably IL-6, Stat3 activation in myeloid cellsactivates Stat3 in tumor cells, enhancing tumor cell proliferation andsurvival (Bollrath et al., 2009; Grivennikov et al., 2009; Lee et al.,2009; Wang et al., 2009).

It is desired to develop new molecules and methods for the treatment ofcancer and other diseases, including new molecules and methods fortreatment that involve pathways within cells that modulate the disease,such as the Stat3 pathway.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for thetreatment of diseases, including cancer, infectious diseases andautoimmune diseases. The present invention also relates to methods andcompositions for improving immune function. The present inventionrelates to blocking Stat3, either through genetic knockout, Stat3small-molecule inhibitor, or Stat3 siRNA, which drastically improves theimmune responses induced by CpG.

The present invention relates to multifunctional molecules that arecapable of being delivered to cells of interest. The multifunctionalmolecules incorporate an activation element together with a therapeuticelement, e.g., a Stat3 blocking element. The multifunctional moleculesare capable of being delivered to specific cells of interest including,but not limited to, dendritic cells. These molecules are capable oftreating diseases, including cancer, infectious diseases and autoimmunediseases. More particularly, the present invention is related tochimeric molecules consisting of an active oligonucleotide, such asToll-like receptor (TLR) ligands, and an active agent, such as doublestranded RNA, such as siRNA or activating RNA. Such chimeric moleculesare taken up and internalized by immune cells and malignant cells,allowing actions of both the TLR ligand and the active agent. Morespecifically, the present invention relates to specific chimericmolecules that are useful for the treatment of diseases.

In one aspect, the present invention provides a novel molecule for thedelivery of an active agent into cells for the treatment of diseasesincluding, but not limited to cancer, infectious diseases and autoimmunediseases. The novel molecules comprises one or more of a first moietythat directs cell or tissue specific delivery of the novel moleculelinked to one or more of a second moiety that is an active agent usefulfor treating cancer or other diseases. The moieties can be linkedtogether directly or they can be linked together indirectly through alinker. In one embodiment, the novel molecule comprises two moieties asone molecule that is multifunctional. For example, a TLR ligand and ansiRNA are made into one molecule for delivery, immune stimulation andblocking immunosuppressive elements, such as Stat3, and/or oncogeniceffects, such as caused by Stat3. In another embodiment, the novelmolecule comprises multifunctional moieties attached to a linker, suchthat it can contain a multitude of moieties. In another embodiment, thelinker is bifunctional producing a molecule of the structure A-X-B,where X is a linker, one of A and B is a moiety that is capable ofdelivering the molecule to cells of interest and the other one of A andB is an active agent useful for treating the cancer or other disease. Aand/or B may also be subject to further linking. In another embodiment,the linker is multifunctional, producing a molecule having more than twomoieties. In one embodiment, using as an example a quadrifunctionalform, such a molecule can have the structure

where X is a linker with four binding sites, one or more of A, B, Y andZ is a moiety that is capable of delivering the molecule to cells ofinterest and the others are an active agent useful for treating thecancer or other disease. In one embodiment, the active agent is a doublestranded RNA molecule that either downregulates gene expression, such asan siRNA molecule, or activates gene expression, such as an activatingRNA molecule. In another embodiment, the active agent is a smallmolecule drug or peptide. In one embodiment, the delivery moiety is aligand for a toll-like receptor (such as oligonucleotides describedherein). In another embodiment, the delivery moiety is anothercell-specific ligand (including, but not limited to, aptamers).

The binding sites on a linker may be specific for each type of moiety tobe linked, for example a linker with a structure that has one regioncapable of likening to an oligonucleotide and another region capable ofbinding to a peptide. Other variations of structure can be proposed byutilizing structures and linkers that promote branching, circularizationor linearization of the molecules, including combinations thereof. Anyelement of a multimeric molecule, including the linker, may also haveadditional functional properties such as being a substrate for chemicalreactions, including enzyme catalyzed reactions, lability inenvironmental conditions such as oxygen tension, pH, ionic conditions.In addition, any element of a multimeric molecule, including linkers mayalso include labels to promote detection—using active or passivedetection of electromagnetic emissions (e.g. optical, ultraviolet,infra-red), radioactivity, magnetic resonance or ability to be cleavedor catalyse a reaction. Many means are available to promote thisincluding use of fluorochromes, quantum dots, dyes, inherent physicalchemical properties structures such as spectral absorbance or emissioncharacteristics magnetic resonance enhancers, and radioisotopes.

In a second aspect, the present invention provides a method for thetreatment of diseases (including, but not limited to, cancer, infectiousdiseases, autoimmune diseases, diseases due to excessive angiogenesisand diseases that can benefit from increased angiogenesis) whichcomprises using the novel molecules of the present invention. Themolecules of the present invention are administered to patients in needof treatment using conventional pharmaceutical practices.

In a third aspect, the present invention provides active agents that arecapable of acting in the Stat3 pathway which, when taken up by the cellsof interest, results in the treatment of diseases including, but notlimited to, cancer, infectious diseases and autoimmune diseases.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 a-1 i show that ablating Stat3 drastically improves TLR ligandinduced antitumor effects which is caused by immune activation. Micewith Stat3^(+/+) and Stat3^(−/−) hematopoietic cells were challengedwith B16 melanoma tumors (s. c.) and treated with a single peritumoralinjection of 5 μg CpG ODNs. FIG. 1 a: Changes in tumor volume within 3days post-CpG treatment. FIG. 1 b: Results from two independentexperiments with either smaller (10 mm³) or larger (70 mm³) averagetumor sizes at the time of CpG ODN injection (n=4). FIGS. 1 c and 1 d:Blocking Stat3 signaling in immune cells leads to CpG-induced tumoreradication and improved survival, which is in part mediated through CD4and CD8 T cells. Mice with B16 tumors were treated with a singleperitumoral injection of CpG ODNs. Depleting antibodies against CD4⁺ andCD8⁺ T cells were given to the indicated groups of mice. Rat IgGantibody was used as a control. Shown are the results representative ofthree independent experiments. FIG. 1 e: Stat3 ablation enhancesTLR9-mediated DC maturation within tumor-draining lymph nodes in vivo.The phenotypic analysis of CD11c⁺ DCs residing in tumor-draining lymphnodes of Stat3^(+/+) and Stat3^(−/−) mice 48 h post-CpG injection. Thematuration of CD11c⁺ DCs is increased by Stat3 ablation as shown by agreater percentage of double-positive MHC class II^(hi) and CD86^(hi)DCs (upper panels), as well as higher expression of costimulatorymolecules CD80 and CD40 on DCs (lower panels). Shown are representativeresults of FACS analysis from one of three independent experiments with3-4 mice per group. FIG. 1 f: Expression of proinflammatory mediators isstrongly upregulated in DCs isolated form CpG-treated tumors. Upperpanel—both p35 and p40 subunits of IL-12, RANTES and IL-6 is upregulatedin Stat3^(−/−) DCs in vivo 18 hrs after CpG treatment. Shown are theresults of real-time PCR analysis of gene expression in CD11c⁺ cellsisolated from tumor-draining lymph nodes. Lower panel—enhanced secretionof proinflammatory cytokines and chemokines by tumor-infiltratingStat3^(−/−) DCs within 48 h post-CpG injection. Cytokine and chemokineexpression was analyzed using antibody arrays in supernatants collectedfrom cultured tumor-infiltrating DCs isolated from Stat3^(+/+) andStat3^(−/−) mice without or after CpG treatment. FIG. 1 g: CD8⁺lymphocyte subsets in tumor-draining lymph nodes of Stat3^(−/−) miceshow increased activation 24 h after CpG ODN injection. The expressionof the early lymphocyte activation marker CD69 was analyzed by flowcytometry on CD8⁺ T cells. Results shown represent one of threeindependent experiments using lymph node cell suspensions from 3-4 miceper group. FIG. 1 h: Stat3^(−/−) mice mount stronger response against anendogenous B16 tumor-antigen than their Stat3^(+/+) counterparts,following treatment with CpG ODN. IFN-γ production in T cells derivedfrom tumor-draining lymph node was assessed by ELISPOT assay. Data shownare mean numbers of p15E-specific IFNγ-producing spots from one of twoseparate experiments with cells pooled from four separate animals pergroup analyzed. FIG. 1 i: FIG. 1 i: Blocking Stat3 using asmall-molecule Stat3 inhibitor drastically improves CpG antitumoreffects. Top panel: growth of B16 tumor is significantly inhibited whenperitumoral CpG ODNs treatment is combined with systemic inhibition ofStat3 activity by a Stat3 inhibitor, CPA7. Mice with established tumors(average diameter 5-8 mm) were treated with CPA7, followed byperitumoral CpG injection a day later. The treatment was repeated twiceweekly. Bottom panel: local CpG treatment promotes concomitant antitumorimmunity when Stat3 activity is systemically suppressed. Mice survivingafter primary tumor challenge were injected with the same tumor cells asthe primary tumor challenges into the opposite flanks. Shown are theresults representative of three independent experiments; n=10 for eachexperiment.

FIGS. 2 a-2 f show that Stat3 siRNA fusion construct mediates Stat3silencing in TLR9⁺ dendritic cells and macrophages. FIG. 2 a: Upperpanel: Sequence of the CpG1668-Stat3 siRNA construct: deoxynucleotides(left portion of molecule) in CpG1668 sequence (SEQ ID NO:1) werephosphothioated and connected through linker (7 units of C3 spacer) tothe antisense strand of a Stat3 siRNA (right portion of molecule;antisense strand: SEQ ID NO:2; sense strand: SEQ IED NO:3)). Lowerpanel: CpG-Stat3 siRNA is processed to active 21-mer siRNA byrecombinant Dicer in vitro. Various double stranded siRNAs wereincubated with 1 U of recombinant Dicer for 1 h at 37° C. and thenvisualized on polyacrylamide gel through SYBRGold staining FIG. 2 b:Left panels: splenocytes were incubated for 24 h with two concentrationsof CpG-linked mouse Stat3 siRNA (CpG-Stat3 siRNA, three upper panels) orunconjugated mouse Stat3 siRNA labeled with fluorescein (bottom panel).Percentage of fluorescein-positive DCs, macrophages, granulocytes, Bcells and T cells was assessed by FACS analysis. Splenic CD11c⁺ DCsexpress high levels of TLR9. Intracellular staining of TLR9 as shown infixed splenic DCs by flow cytometry. FIG. 2 c: CpG-Stat3 siRNA-FITC isquickly internalized by dendritic cells in the absence of transfectionreagents. The uptake by DC2.4 cells is analyzed by flow cytometry (upperpanel) and confocal microscopy (lower panels) after incubation times asindicated. FIG. 2 d: Internalized CpG-Stat3 siRNA colocalizes with TLR9(two upper rows) and transiently interacts with Dicer (two lower rows)as shown by confocal microscopy. DC2.4 cells were incubated with 500pmol/ml of CpG-Stat3siRNA for times as indicated. Shown are confocalmicroscopy images; green: CpG-Stat3 siRNA-FITC, red—TLR9 or Dicer,blue—nuclear staining with Hoechst. FIG. 2 e: Treatment withCpG-Stat3siRNA leads to silencing of Stat3 expression in DC2.4 cells.Cells were treated for 24 hrs with 1 μM CpG-Stat3 siRNA or CpG-scrambledRNA. Shown are the results of real-time PCR for Stat3, normalized toGAPDH levels. The level of Stat3 expression in CpG-scrambled RNA sampleis set as 100%. FIG. 2 f: Stat3 DNA-binding is reduced following 48 h ofincubation with CpG-Stat3 siRNA but not with CpG-scrambled RNA.

FIGS. 3 a-3 h show that treatment with CpG-Stat3 siRNA leads toantitumor effects in vivo. FIG. 3 a: In vivo uptake of intratumorallyinjected CpG-Stat3 siRNA by myeloid cells. Upper panel:immunofluorescent imaging on frozen tumor and lymph node tissue sections6 h after CpG-construct injection. Green: FITC-labeled CpG-Stat3 siRNA,red: staining with anti-CD11b-specific antibody, blue: nuclear stainingwith Hoechst. Lower panel: intravital two-photon microscopy ontumor-draining lymph node within 1 h after intratumoral injection ofFITC-labeled CpG-Stat3 siRNA (green), blood vessels: red, nuclei: blue;top right panel: close-up of the lymph node tissue to visualizeincreased number of FITC-positive cells entering the lymph node, bottomright panel: intracellular distribution of FITC-labeled CpG-Stat3 siRNA.FIG. 3 b: Local treatment with CpG-Stat3 siRNA reduces Stat3 expressionin DCs within tumor draining lymph nodes. Total RNA was isolated fromtumor-draining lymph node DCs and analyzed by real-time PCR. FIG. 3 c:B16 tumor growth is inhibited by local treatment with CpG-Stat3 siRNA.Mice with subcutaneously growing tumors were treated by repeatedperitumoral injections of 14 μg CpG-Stat3 siRNA, GpC-Stat3 siRNA,CpG-scrambled RNA or combination of equimolar amounts of uncoupled CpGand Stat3 siRNA every second day, starting six days after challenge with1×10⁵ B16 cells. FIG. 3 d: Right panel: Stat3 expression is reduced bysystemic CpG-Stat3 siRNA treatment in DCs within tumor draining cervicallymph nodes. Shown are results of real-time PCR analysis. FIG. 3 e:Systemic treatment with CpG-Stat3 siRNA reduces the number of B16 tumormetastasis. Mice were injected i.v. with 1×10⁵ B16 cells and treatedwith 14 μg CpG-Stat3 siRNA or CpG-scrambled RNA injections every secondday starting from two days post-challenge. Lung colonies were enumerated15 days later when mice become moribund. Significant differences betweenmean numbers±SEM, of CpG-Stat3 siRNA or CpG-scrambled RNA-treated miceare indicated (right panel). Representative picture of lung excised frommice inoculated and treated as described above (left panel). FIGS. 3 fand 3 g: Stat3 inhibition promotes DC maturation (FIG. 3 f) andincreases ratio of effector to regulatory T cells within tumor tissue(FIG. 3 g). Single cell suspensions prepared from tumor-draining lymphnodes (FIG. 3 f) or tumors (FIG. 3 g) treated with peritumoralinjections of CpG-Stat3 siRNA or CpG-scrambled RNA as described in 3 a,were analyzed by flow cytometry. FIG. 3 h: Local treatments withCpG-Stat3 siRNA lead to increased tumor infiltration by CD8⁺ T cells(left), and generate tumor antigen-specific CD8+ T cell immune responsesas measured by TRP-2 specific IFN-γ ELISPOT (right).

FIGS. 4 a-4 d show that CpG(D19)-STAT3 siRNA allows for targeting STAT3in human monocytes and monocyte-derived DCs. CpG(D19)-STAT3siRNA isinternalized specifically by CD14⁺ monocytes from human PBMCs (FIG. 4 a)and cultured monocyte-derived DCs in dose- (FIG. 4 b) and time-dependentmanner (FIG. 4 c) as measured by flow cytometry. FIG. 4 d: STAT3silencing in monocyte-derived DCs. Enriched CD14⁺ monocytes werecultured for 6 days in the presence of GM-CSF and IL-4 with the additionof fluorescein-labeled CpG(D19)-STAT3 siRNA or CpG-scrambled RNAcontrol. The expression of STAT3 was estimated by real-time PCR on totalRNA isolated on day 6.

FIGS. 5 a-5 e show that CpG-STAT3 siRNA mediates siRNA delivery intohuman and mouse tumor cells of hematopoietic origin. FIG. 5 a:Dose-dependent uptake of FITC-labeled CpG-STAT3 siRNA by human L540Hodgkin's lymphoma cells after overnight incubation. FIG. 5 b: CpGsiRNAinternalization by human different types of lymphoma cells. Cells ofeach type were incubated overnight with 500 nM FITC-labeled CpG-STAT3siRNA and analyzed with flow cytometry. FIG. 5 c: MCP11 cellsinternalize FITC-labeled CpG-Stat3 siRNA in a dose-dependent manner, asshown by flow cytometry after 24 h incubation. FIG. 5 d: Stat3 silencingin MPC 11 cells treated with 100 nM CpG-Stat3 siRNA for 24 h, asmeasured by real-time PCR. Con, control scrambled siRNA, siRNA=mouseStat3siRNA. FIG. 5 e: MCP11 cells accumulate in the G₂M phase of cellcycle after 48 h incubation with CpG-Stat3 siRNA as measured by flowcytometry after propidium iodide staining.

FIGS. 6 a-6 c show that targeting Stat3 by CpG-Stat3 siRNA leads toantitumor effects against MPC11 multiple myeloma. FIG. 6 a: In vivotreatment with CpG-Stat3 siRNA results in immune activation and tumorgrowth inhibition. Mice bearing large MCP11 tumors (10-13 mm indiameter) were injected intratumorally with 0.78 nmole of CpG-Stat3siRNAor CpG-scrRNA, followed by two more times every second day. FIG. 6 b:Increased percentage of DCs in tumor-draining lymph nodes afterCpG-Stat3siRNA treatment. FIG. 6 c: CD40 and CD86 expression onactivated DCs in tumor-draining lymph nodes as measured by flowcytometry in CpG-siStat3 (red) or CpG-scrRNA (blue) injected micecomparing to untreated controls.

FIG. 7 shows selection of the most effective human and mouse STAT3 siRNAsequences. More than 50 double-stranded oligoribonucleotides (27mer,Dicer substrate) with potential STAT3 siRNA sequences were tested inhuman A2058 or mouse B16 melanoma cells. STAT3 silencing was assessed byquantitative real-time PCR, 24 h after transfection, and normalized toGAPDH expression. Control=scrambled siRNA, arrows indicate the mostpotent STAT3 siRNAs. The sequences of the optimal Stat3 siRNAs areshown. Human sense strand is SEQ ID NO:4; human antisense strand is SEQID NO:5; mouse sense strand is SEQ ID NO:3; mouse antisense strand isSEQ ID NO:2.

FIG. 8 shows TLR ligand-linker-Stat3siRNA sequences. Mouse Stat3 siRNA(SS): SEQ ID NO:3; CpG1668:SEQ ID NO:1; mouse Stat3 siRNA (AS): SEQ IDNO:2; GpC: SEQ ID NO:6; human Stat3 siRNA (SS): SEQ ID NO:4; CpG(D19):SEQ ID NO:7; human Stat3 siRNA (AS): SEQ ID NO:3; scrambled RNA (SS):SEQ ID NO:8; scrambled RNA (AS): SEQ ID NO:9).

FIG. 9 shows that a double stranded RNA with sequences complimentary tosequences of the mouse Edg1 gene promoter region are able to activateEdg1 expression in vitro. The Edg1 double stranded RNA when transfectedinto cells (both 3T3 fibroblasts and B 16 tumor cells) induces strongtranscription of the Edg1 gene, as determined by real-time PCR.

FIG. 10 shows that the activating RNA is active for at least three weeksin living animals. Tumor cells transfected with the activating RNA forEdg1 promoter, when implanted into mice, maintain high levels of Edg1expression for at least 3 weeks, as determined by analyzing tumors forEdg1 expression using real-time PCR at three weeks after tumorimplantation.

FIGS. 11 a-11 d show the structure and function of the CpG-Stat3 siRNAconjugate. FIG. 11 a: Sequence of the CpG-linked mouse Stat3 siRNAconjugate (CpG1668-Stat3 siRNA): CpG1668 sequence (deoxyribonucleotides;SEQ ID NO:1) were phosphothioated and connected through a carbon linker(6 of C3 units) to the antisense strand of Stat3 siRNA (ribonucleotidesin the upper strand (SEQ ID NO:2); ribonucleotides in the lower strandexcept AA on the 3′ end which are deoxyribonucleotides (SEQ ID NO:3).FIG. 11 b: CpG-siRNA has similar immunostimulatory activity compared touncoupled CpG ODN, as indicated by increased expression of costimulatorymolecules, CD40 and CD80, on primary splenic DCs after 24 h incubationwith or without ODNs; splenocytes were pooled from 2-3 mice and theexperiment was done twice with similar results. FIG. 11 c: LinkedCpG-Stat3 siRNA is processed to active 21mer siRNA by recombinant Dicerin vitro. Comparable processing of conjugated CpG-Stat3 siRNA moleculesand uncoupled Stat3 siRNA visualized on polyacrylamide gel throughSYBRGold staining; position of the 21/21mer and the remaining part ofthe molecule (CpG plus carbon linker) are indicated. FIG. 11 d: Stat3siRNA linked to CpG ODN retains the ability to mediate RNA interference.B16 cells were transfected using lipofectamine reagent with CpG-linkeddsRNAs or unconjugated dsRNAs in the presence of 15 nM CpG ODN asindicated. Stat3 gene silencing effects were evaluated by western blotanalysis.

FIGS. 12 a-12 d show the relative immunostimulatory properties ofvarious oligonucleotide sequences used in the study. FIGS. 12 a-12 c:Freshly isolated splenocytes (pooled from three C57BL/6 mice) werecultured for 24 h in the presence of LPS (0.5 μg/ml) or variousoligonucleotides (500 nM of each) as indicated. The secretion ofproinflammatory mediators including IL-6 (FIG. 12 a), TNFα (FIG. 12 b)and IFNγ (FIG. 12 c) into culture media was assessed using ELISA. FIG.12 d: Immunostimulatory effects of various TLR agonists as measuredusing SEAP reporter gene assay. RAW-Blue™ cells (InvivoGen) wereincubated for 24 h with LPS (5 μg/ml) or 500 nM of variousoligonucleotides as indicated. The level of immune activation wasassessed based on NF-κB/AP1-dependent induction of SEAP expressionmeasured calorimetrically. Shown are representative results of twoindependent experiments analyzed in triplicates±SEM. This figure showsthat linking siRNA to CpG does not create non-specific immune response.

FIGS. 13 a-13 f show CpG-Stat3 siRNA uptake and gene silencing in vitro.FIG. 13 a: Targeted delivery: splenocytes derived form 2-3 mice wereincubated for 3 h with various concentrations of CpG-Stat3 siRNA (toptwo columns) or for 24 h with unconjugated Stat3 siRNA labeled withfluorescein (bottom right panel) in the absence of any transfectionreagents. Percentage of fluorescein-positive CD11c⁺B220⁻non-plasmacytoid (mDCs) and CD11c⁺B220⁺ plasmacytoid (pDCs) DCs,F4/80⁺Gr1⁻ macrophages (MACs), B220⁺CD11c⁻ B cells, Gr1⁺F4/80⁻granulocytes and CD3⁺ T cells was assessed by FACS analysis (see alsoTable 1). Splenic CD11c⁺ DCs express high levels of TLR9. Intracellularstaining of TLR9 as shown in fixed splenic DCs by flow cytometry (bottomright panel). Similar results were obtained in two independentexperiments using splenocytes pooled from 3 mice. FIG. 13 b: Kinetics ofCpG-siRNA internalization: CpG-Stat3 siRNA-FITC is quickly internalizedby dendritic cells in the absence of transfection reagents. The uptakeby DC2.4 cells is analyzed by flow cytometry (top row) and confocalmicroscopy (two lower rows) after incubation with CpG-Stat3 siRNA-FITCat the concentration of 500 nM for indicated times (two upper rows) orafter 1 h incubation concentrations as indicated (bottom row); shown areresults representative for 3 independent experiments. FIG. 13 c:Internalized CpG-Stat3 siRNA colocalizes with TLR9 (two upper rows) andtransiently interacts with Dicer (two lower rows) as shown by confocalmicroscopy. DC2.4 cells were incubated with 500 nM of CpG-Stat3 siRNAfor indicated times. Shown are confocal microscopy images;green—CpG-Stat3 siRNA-FITC, red—immunofluorescent detection ofendogenous TLR9 or Dicer, blue—nuclear staining with DAPI. All confocalimaging studies were performed at least thrice with similar results andthe images acquired were characteristic for the majority of analyzedcells (FIG. 16). FIG. 13 d: Dose-dependent gene silencing effects ofCpG-Stat3 siRNA, comparing to GpC-Stat3 siRNA at the highest dose, asdetermined by quantitative real-time PCR in DC2.4 cells. Shown are theresults of real-time PCR for Stat3, normalized to GAPDH expressionlevels. The level of Stat3 expression in CpG-scrambled RNA sample is setas 100%. Shown are means±SEM from three independent experiments analyzedin duplicates. FIG. 13 e: Stat3 silencing is impaired in TLR9-deficientprimary myeloid cells (top panel) and dendritic cells (bottom panel).Shown are results of two independent experiments, analyzed intriplicates by real-time PCR; means±SEM. FIG. 13 f: Stat3 DNA-binding isreduced following 48 h incubation of DC2.4 cells with CpG-Stat3 siRNA,relative to CpG-scrambled RNA. Shown are results of electrophoreticmobility gel-shift assay using radiolabeled probe specifically bound byStat3 and Stat1 in one of three independent experiments. Positions ofStat dimers are indicated.

FIG. 14 shows the gating of various immune cell subsets for the analysisof in vitro CpG-Stat3 siRNA uptake. FACS analysis was performed onsingle-cell suspensions of splenocytes prepared as described in thelegend to FIG. 13 b. The percentages of FITC-positive cells shown inFIG. 13 b and Table 1 were assessed in immune cell subtypes gated asindicated.

FIG. 15 shows a comparison of internalization kinetics of CpG ODN, siRNAand CpG-siRNA conjugate. The uptake of FITC-labeled molecules by DC2.4cells was analyzed by flow cytometry after incubation at 500 nM forindicated times.

FIG. 16 shows colocalization of FITC-labeled CpG-Stat3 siRNA with TLR9(top panels) and with Dicer (bottom panels) as shown by confocalmicroscopy. DC2.4 cells were incubated with 500 nM of CpG-Stat3 siRNAfor 1 h. Shown are confocal microscopy images at lower magnification tovisualize similar colocalization pattern in the majority of analyzedcells; green—CpG-Stat3 siRNA-FITC(C/S-FITC), red—immunofluorescentdetection of endogenous TLR9 or Dicer, blue—nuclear staining withHoechst. All confocal imaging studies were performed at least twice withsimilar results. Scale bar=10 μm.

FIG. 17 shows that TLR9 is not required for uptake of FITC-labeledCpG-Stat3 siRNA. Cultured bone marrow-derived DCs (day 9) were incubatedfor 1 h with 500 nM CpG-Stat3siRNA labeled with FITC. Shown arepercentages of fluorescein-positive CD11c⁺ cells as assessed by FACS.

FIGS. 18 a-18 b show CpG-siRNA uptake and gene silencing effects in A20B cell lymphoma cells. FIG. 18 a: A20 cells were cultured in thepresence of the chimeric constructs for 24 h at indicatedconcentrations. FIG. 18 b: CpG-Luc siRNA silences gene expression asshown by reduction in luciferase activity. CpG-Luc siRNA andCpG-scrambled RNA were added to Luc-A20 cells (100 nM). Shown areaverages±SEM, n=5.

FIG. 19 shows the biodistribution of systemically injected CpG-Stat3siRNA. Mice were sacrificed 3 h after retroorbital venous injection of100 μg FITC-labeled CpG-Stat3 siRNA. Harvested tissues wereenzymatically dispersed into single cell suspension, enriched formononuclear cells and analyzed by FACS for the presence of variousimmune cell subsets as indicated. Shown are representative results oftwo independent experiments using 2-3 mice analyzed individually. Thisfigure shows that the systemic delivery of siRNA by CpG-siRNA constructefficiently targets myeloid cells in liver, kidney and lung.

FIGS. 20 a-20 e show that treatment with CpG-Stat3 siRNA leads tocell-specific gene silencing in vivo. FIG. 20 a: In vivo uptake ofintratumorally injected CpG-Stat3 siRNA by myeloid cells. Shown isimmunofluorescent staining of frozen tumor tissue section 6 h afterinjection of CpG-siRNA conjugate. Green: FITC-labeled CpG-Stat3 siRNA;red: myeloid cells stained with anti-CD11b antibodies: blue: nuclearstaining with Hoechst. FIG. 20 b: Left panel—intravital two-photonmicroscopy on tumor-draining lymph node at 1 h after intratumoralinjection of FITC-labeled CpG-Stat3 siRNA (green); blood vessels stainedwith dextran-rhodamine (red); Hoechst-stained nuclei (blue). Top rightpanel: close-up of the lymph node tissue to visualize increased numberof FITC-positive cells entering the lymph node; bottom right panel:intracellular distribution of FITC-labeled CpG-Stat3 siRNA. Resultsrepresentative for two independent experiments using 2 mice perexperiment are shown. FIGS. 20 c and 20 d: Repeated local peritumoraltreatment with CpG-Stat3 siRNA significantly reduces Stat3 mRNA andprotein in immune cells within tumor draining lymph nodes. Total RNA andprotein were isolated from CD11c⁺ DCs, CD19⁺ B cells and CD11b⁺c⁻myeloid cells accumulated in tumor-draining inguinal lymph nodes usingcells pooled from 4-9 mice. FIG. 20 c: Shown are combined results ofquantitative real-time PCR analysis from 3-4 independent experiments±SEMcomparing Stat3 expression levels in CpG-Stat3 siRNA-treated mice inrelation to control CpG-Luc siRNA set as 100%. FIG. 20 d: Stat3activation and protein levels are reduced by CpG-Stat3 siRNA but not bycontrol CpG-Luc siRNA conjugates. Representative results of Western blotanalysis for tyrosine-phosphorylated or total Stat3 and β-actin from oneof two independent experiments are shown. FIG. 20 e: Two weeks after B16tumor challenge, luciferase-overexpressing mice were injectedperitumorally with CpG-Luc siRNA or CpG-scrambled RNA every day for atotal of three injections. The level of luciferase activity was assessedin CD11b⁺ and CD4⁺ cells isolated from tumor-draining lymph nodes; shownare representative results from one of 3 independent experiments using 3mice/group.

FIGS. 21 a-21 b show the kinetics of CpG-Stat3 siRNA uptake in vivofollowing intratumoral injection. Mice were sacrificed after i.t.injection of 20 μg FITC-labeled CpG-Stat3 siRNA at indicated times.Single-cell suspensions of tumors (FIG. 21 a) and tumor-draining lymphnodes (FIG. 21 b) were enriched for viable mononuclear cells andanalyzed by FACS for the presence of F4/80⁺CD11c⁻ macrophages and CD11c⁺DCs. Shown are representative results of two independent experimentsusing 2-3 mice analyzed individually. This figure shows that local tumortreatment allows CpG-siRNA to enter macrophages and dendritic cells.

FIG. 22 shows the in vivo uptake of intratumorally injected CpG-Stat3siRNA by myeloid cells. Intravital two-photon microscopy ontumor-draining and contra-lateral lymph nodes at 1 h after singleintratumoral injection of 20 μg FITC-labeled CpG-Stat3siRNA (green)together with intravenously injected Hoechst 33342 for nuclear staining(blue). For tumor-draining lymph node, the overlay image was split intogreen (upper right panel) and blue (lower right panel) channels tovisualize better cellular localization of the injected CpG-Stat3siRNA.

FIGS. 23 a and 23 b show the local treatment with CpG-Stat3 siRNAreduces Stat3 expression within total tumor-draining lymph nodes. Micewith subcutaneous B16 tumors were treated by repeated peritumoralinjections using various CpG-RNAs or PBS alone as indicated, everysecond day, starting six days after challenge with 1×10⁵ B16 cells using7 mice/group. Single cell suspensions prepared from pooledtumor-draining lymph nodes were used for further analyses of Stat3expression. FIG. 23 a: Local treatment using CpG-Stat3 siRNA leads toStat3 silencing in tumor-draining lymph nodes. Shown are the results ofreal-time PCR for Stat3, normalized to GAPDH levels. The level of Stat3expression in control PBS-treated sample is set as 100%. FIG. 23 b:Levels of Stat3 protein in total tumor-draining lymph node cells arereduced by CpG-Stat3 siRNA but not control conjugates, CpG-scrambled RNAand CpG-Luc siRNA. Representative results of Western blot analysis fromone of two independent experiments are shown.

FIGS. 24 a-24 g show that local treatment with CpG-Stat3 siRNA inhibitstumor growth. FIG. 24 a: Mice with subcutaneous B16 tumors were treatedby peritumoral injections of CpG-Stat3 siRNA, GpC-Stat3 siRNA,CpG-scrambled RNA, combination of equimolar amounts of uncoupled CpG andStat3 siRNA or PBS only every other day, starting six days afterchallenge with 1×10⁵ B16 cells, n=5-6. Statistically significantdifferences between CpG-Stat3 siRNA- and CpG-scrambled RNA-treatedgroups are indicated by asterisks. Similar results were reproduced inthree independent experiments. FIG. 24 b: Tumor growth inhibition byCpG-Stat3 siRNA depends on NK cell- and T cell-mediated immunity. Micewith established B16 tumors were depleted of NK cell or CD4/CD8lymphocytes prior to the repeated treatment with CpG-Stat3 siRNA everyother day; shown are means±SEM, P<0.0001 (from two-way ANOVA test). FIG.24 c and FIG. 24 d: Local treatment with CpG-Stat3 siRNA reduces growthof other tumor models independently of genetic background. C4 melanomacells (FIG. 24 c) and CT26 colon carcinoma cells (FIG. 24 d) wereinjected s.c. into C3H or BALB/c mice, respectively. Mice withestablished tumors were treated by peritumoral injections of CpG-Stat3siRNA, CpG-Luc siRNA, CpG alone or PBS every other day, starting seven(C4, CT26) days after challenge with 1×10⁵ tumor cells. Statisticallysignificant differences between CpG-Stat3 siRNA- and CpG-LucsiRNA-treated groups are indicated by asterisks. FIG. 24 e: C57BL/6.CEAmice were challenged s.c. with 1×10⁵ of MC38.CEA cells and treated asdescribed above using CpG-Stat3 siRNA (left panel) or CpG-Luc siRNA(right panel) starting from day 11. Shown are tumor growth curves forboth groups with statistically significant differences indicated byasterisks; P<0.0001 by two-way ANOVA (n=4 for each group). FIG. 24 f:Systemic treatment using CpG-Stat3 siRNA reduces Stat3 expression in DCswithin tumor-draining cervical lymph nodes. Samples pooled from 6mice/group were analyzed by real-time PCR. Shown is the average level ofStat3 expression in CpG-Stat3 siRNA-treated mice from one of twoindependent experiments analyzed in triplicates±SEM in relation tocontrol CpG-scrambled RNA set as 100%. FIG. 24 g: Systemic treatmentwith CpG-Stat3 siRNA reduces the number of B16 tumor metastasis. Micewere i.v. injected with B16 cells and treated with CpG-Stat3 siRNA orCpG-scrambled RNA injections every other day starting from two days posttumor challenge. Lung colonies were enumerated 15 days later whencontrol mice become moribund. Shown are mean numbers of colonies±SEM(n=7), analyzed for statistical significance by two-way ANOVA test;P=0.0054. Representative photos of lung excised from mice inoculated andtreated as described above. The in vivo data are representative of twoindependent experiments.

FIG. 25 shows augmented cell apoptosis within B16 tumors followingperitumoral treatment with CpG-Stat3siRNA. Frozen sections prepared fromB16 tumors injected 3 times using PBS, CpG, or CpG-siRNA conjugates,were analyzed by immunofluorescence using antibodies specific to activecaspase-3 to detect apoptosis (red) and counterstained with Hoechst(blue) for visualization of nuclei. Shown are representative resultsfrom two independent experiments using samples isolated from 4individual mice; original magnification, ×100.

FIGS. 26 a-26 c show in vivo administration of CpG-Stat3 siRNA inducesproinflammatory cytokine expression and activates innate immunity. FIG.26 a: Immunostimulatory cytokine/chemokine gene expression was analyzedin DCs enriched from tumor-draining lymph node cell suspensions pooledfrom 4-10 mice, prepared after 3 peritumoral injections of CpG-Stat3siRNA or CpG-Luc siRNA. Data from quantitative real-time PCRs run intriplicates were normalized to GAPDH expression. The averaged resultsfrom 4 independent in vivo experiments were combined and analyzed forstatistical significance using unpaired t-test with unequal variance.Shown are mean values±SEM; CpG-Luc siRNA was set as a baseline (100%).FIG. 26 b: Frozen sections of tumor tissues isolated from mice aftertreatments as indicated, were stained with antibodies specific toneutrophils (green) and activated caspase-3 (red) and analyzed byfluorescent microscopy. Shown are results of two independentexperiments; original magnification: ×100. FIG. 26 c: Single cellsuspensions prepared from tumors pooled from 3-6 mice were analyzed byflow cytometry for the presence of Gr1⁺CD11b⁻ neutrophils. Shown aremeans±SEM combined from three independent experiments.

FIG. 27 shows reduction of immature DCs in the tumor draining lymphnodes. B16 tumor bearing mice were treated peritumorally withCpG-Stat3siRNA or CpG-scrambled RNA as described in FIG. 20 d. Shown areresults of flow cytometric analyses performed on single cell suspensionsprepared from tumor-draining lymph nodes pooled from 5-6 mice; thepercentages of DCs with low expression of MHC class II or co-stimulatorymolecules are indicated.

FIGS. 28 a and 28 b show targeting Stat3 using CpG-siRNA augments innateand adaptive antitumor immunity. Effects of in vivo CpG-siRNA treatmenton immune cell populations within tumor. Single cell suspensionsprepared from tumors pooled from 3-6 mice were analyzed by flowcytometry for the presence of CD4⁺ (FIG. 28 a), CD4⁺FoxP3⁺ (FIG. 28 b)and CD8⁺ (FIG. 28 c). Shown are means±SEM from combined threeindependent experiments and representative dot plots (left panels in(FIG. 28 b, FIG. 28 c). FIG. 28 d: Local treatments with CpG-Stat3 siRNAgenerate tumor antigen-specific immune responses as measured by ELISPOT.IFNγ ELISPOT assays were performed using cell suspensions prepared fromfour pooled tumor-draining lymph nodes per each treatment group asdescribed in FIG. 3 d; presented are the results form one of twoindependent experiments. Bars represent average numbers of TRP2-specificdots±SEM from triplicate samples; P-values from one-way ANOVA test forstatistical significance are indicated.

FIGS. 29 a-29 c show Stat3 silencing and antitumor responses induced byalternative sequences of Stat3 siRNA conjugated with CpG. FIG. 29 a: NewStat3 sequences of single stranded constructs (deoxynucleotides areshown underlined). Mouse Stat3 siRNA #2 (SS): SEQ ID NO:12;CpG1668-mouse Stat3 siRNA #2 (AS): SEQ ID NO:1-linker-SEQ ID NO:13;mouse Stat3 siRNA #3 (SS): SEQ ID NO:14; CpG1668-mouse Stat3 siRNA #2(AS): SEQ ID NO:1-linker-SEQ ID NO:15. FIG. 29 b: New CpG-Stat3 siRNAsstimulate antitumor responses in vivo. Mice with established s.c. B16tumors (average diameter 10 mm) were treated by peritumoral injectionsof CpG-Stat3siRNA in two versions or equimolar amounts of CpG-Luc siRNAevery other day. Shown are means±SEM; P<0.0001 by two-way ANOVA;statistically significant differences between CpG-Stat3 siRNAs- andCpG-Luc siRNA-treated groups indicated by Bonferroniposttest areindicated by asterisks (n=6). FIG. 29 c: Stat3 expression in B cellsfreshly isolated from tumor-draining lymph nodes was assessed usingreal-time PCR, comparing Stat3 expression levels in CpG-Stat3siRNA-treated mice in relation to control CpG-Luc siRNA set as 100%.Shown are means±SEM (n=3); P=0.0018 by one-way ANOVA. This figure showsthe validation of CpG-siRNA approach for cancer immunotherapy by usingadditional siRNAs with different sequences.

FIGS. 30 a and 30 b show that TLR9 is required for silencing effect ofFITC-labeled CpG-Stat3 siRNA by myeloid cells. FIG. 30 a: Freshlyisolated PBMCs were incubated for 1 h with 500 nMCpG-Stat3siRNA labeledwith FITC. Percentages of fluorescein-positive CD11b⁺ myeloid cellsassessed by FACS are indicated. Shown are representative results fromone of two independent experiments. FIG. 30 b: Stat3 silencing isimpaired in TLR9-deficient primary myeloid cells (top panel) anddendritic cells (bottom panel); means±SEM (n=3). This figure shows thatTLR9 is not necessary for uptake but is required for silencing effect ofCpG-Stat3 siRNA by myeloid cells.

FIGS. 31 a-31 e show that CpG-STAT3 siRNA targets human TLR9-positivetumor cells leading to gene silencing and growth inhibition ofxenotransplantmyeloma in mice. FIG. 31 a: The uptake of FITC-labeledCpG-STAT3 siRNA by cultured myeloma cells estimated by flow cytometry.FIG. 31 b: In vivo internalization of CpG-STAT3 siRNA injectedintratumorally. KMS11 tumors grown in NOD/SCID mice were harvested 3 hafter injection of various doses of the conjugate and dispersed intosingle cell suspensions. After removal of CD11b⁺ myeloid cells and CD19⁺B cells, the percentage of FITC+cells was analyzed by FACS. FIGS. 31c-31 e: CpG-STAT3 siRNA in vivo treatment leads to STAT3 gene silencing(FIG. 31 c), tumor cell death (FIG. 31 d) and reduced growth rate ofhuman myeloma tumors in NOD/SCID mice (FIG. 31 e). Tumors were treatedwith daily intratumoral injections of 20 μg CpG-STAT3 siRNA starting 5days after injection of 1×10⁷ of KMS11 myeloma cells (at the averagetumor size 10 mm); P<0.001 (by two-way ANOVA). This figure shows thatthe CpG-siRNA approach effectively silences genes in TLR9⁺ human tumorcells leading to therapeutic antitumor effects in animals.

FIGS. 32 a-32 d show that CpG-STAT3 siRNA approach effectively silencesgenes in TLR9+ human acute myeloid leukemia (AML) cells, leading totherapeutic antitumor effects in xenotransplanted tumor models in mice.FIG. 32 a: NOD/SCID/IL-2Rγnull (NSG) mice were injected i.v. with 107 ofhuman MV4-11 leukemia cells. Four weeks later, mice with engrafted AMLcells were injected i.v. with the 100 μg dose of various CpG(A)-siRNAsdaily for three days. The percentages of viable bone-marrow resident AMLtumor cells after treatment using CpG(A)-Luciferase siRNA (top) andCpG(A)-STAT3 siRNA (bottom) were assessed by FACS using antibodiesspecific for human CD45 expressed on the surface of MV4-11 cells. STAT3gene silencing was assessed in bone marrow-derived AML cells usingquantitative real-time PCR (qPCR) (right graph). FIG. 32 b: CpG(A)-STAT3siRNA in vivo treatment leads to STAT3 gene silencing (left, by qPCR),tumor cell death (middle, by FACS analysis of Annexin V-positive tumorcell suspensions) and reduced growth rate of human myeloma tumors in NSGmice (right). Tumors were treated with daily intratumoral injections of20 μg CpG-STAT3 siRNA starting 4-5 days after injection of 107 of tumorcells (at the average tumor size 10 mm). Blocking of STAT3 in MonoMac6cells (FIG. 32 c) and BCL-XL in MV4-11 AML cells (FIG. 32 d) in vivoinhibits growth of xenotransplanted tumors in NSG mice. The target genesilencing (left graphs in FIG. 32 c, 32 d), tumor cell death (middlegraph in FIG. 32 d) and tumor growth kinetics (right panels) wereassessed as described above. Statistically significant differencesbetween CpG-STAT3 or BCL-XL siRNA- and CpG-Luc RNA-treated groups (fromtwo-way ANOVA test) are indicated by asterisks as described in thelegend for FIG. 34. Shown are the representative results from one of twoindependent experiments (FIG. 32 b) or from single experiments (FIGS. 32a, 32 c, 32 d) using 5-6 mice per each experimental group; means±s.e.m.

FIG. 33 shows the efficacy of in vivo target gene silencing by CpG-STAT3siRNA depends on the CpG ODN sequence. FIG. 33 (top):NOD/SCID/IL-2Rγnull (NSG) mice were injected s.c. with 5×106 of humanMV4-11 leukemia cells. Tumors were treated with two daily intratumoralinjections of 20 μg various CpG-siRNAs as indicated, includingCpG-Luciferase siRNA and CpG-STAT3 siRNA in two versions, conjugated toclass A (D19 ODN) or class B (7909) CpG ODN. The STAT3 gene silencingwas assessed by quantitative real-time PCR (FIG. 33 (top)), while tumorcell death was measured by FACS analysis using Annexin V staining oftumor cell suspensions (FIG. 33 (bottom)). Shown are the representativeresults from a single experiments using 5-6 mice per each experimentalgroup; means±s.e.m.

FIG. 34 shows that the class A ODN-based CpG(D19)-STAT3 siRNA conjugatesinduce production of proinflammatory protein mediators withoutstimulating expression of potentially tumor promoting IL-6, IL-8 orIL-10, which are co-activated by two other CpG-siRNA types. Human PBMCswere incubated for 24 h in the presence of class A—CpG(D19)-STAT3 siRNA,calls B—CpG(7909)-STAT3 siRNA or class C—CpG(2429)-STAT3 siRNAconjugates in concentrations as indicated. Supernatants from culturedPBMCs were analyzed for the production of pro-inflammatory andanti-inflammatory protein mediators using Cytokine Bead Arrays onLuminex platform. Shown are representative results from one of twoindependent experiment performed in triplicates; ND—not detectable.

FIG. 35 shows that the CpG(D19)-STAT3 siRNA does not induce exacerbatedtype I interferon response, in contrast to unconjugated D19 class Aoligodeoxynucleotides. Human PBMCs were incubated for 24 h in thepresence of STAT3 siRNA, CpG(A)-D19, CpG(B)-7909 alone or as CpG-STAT3siRNA conjugates in concentrations as indicated. Supernatants fromcultured PBMCs were analyzed for the IFNα production using Cytokine BeadArray on Luminex platform. Shown are representative results from one oftwo independent experiment performed in triplicates; ND—not detectable.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention belongs.

The present invention relates to methods and compositions for thetreatment of diseases. More particularly, the present invention relatesto multifunctional molecules that are capable of being delivered tocells of interest for the treatment of diseases including, but notlimited to, cancer, infectious diseases and autoimmune diseases. Morespecifically, the present invention relates to specific chimericmolecules that are useful for the treatment of diseases.

In one aspect, the present invention provides a novel molecule for thedelivery of an active agent into cells for the treatment of cancer andother diseases including, but not limited to infectious diseases andautoimmune diseases. The novel molecules comprises one or more of afirst moiety that directs cell or tissue specific delivery of the novelmolecule linked to one or more of a second moiety that is an activeagent useful for treating cancer or other diseases. The moieties can belinked together directly or they can be linked together indirectlythrough a linker. In one embodiment, the novel molecule comprises twomoieties as one molecule that is multifunctional. For example, a TLRligand and an siRNA are made into one molecule for delivery, immunestimulation and blocking immunosuppressive elements, such as Stat3,and/or oncogenic effects, such as caused by Stat3. In anotherembodiment, the novel molecule comprises moieties attached to a linkerthat is multifunctional, such that it can contain a multitude ofmoieties. In another embodiment, the linker is bifunctional producing amolecule of the structure A-X-B, where X is a linker, one of A and B isa moiety that is capable of delivering the molecule to cells of interestand the other one of A and B is an active agent useful for treating thecancer or other disease. In another embodiment the linker is amodification of, or structure present on, either moiety A or B, or both,that results in a binding between the two elements. The binding maybecovalent or non-covalent bonds. In another embodiment, the linker ismultifunctional, for example, quadrifunctional, producing a moleculehaving more than two moieties. In one embodiment, such a molecule canhave the structure

where X is the linker, one or more of A, B, Y and Z is a moiety that iscapable of delivering the molecule to cells of interest and the othersare an active agent useful for treating the cancer or other disease. Thelinker may have any number of other moieties attached to it, and theexamples of having two or four moieties, and their lack of any secondaryextension, for example a modification of Y, is merely for illustrationpurposes and not intended to be limiting.

In one embodiment, the active agent is a double stranded RNA moleculethat either downregulates gene expression, such as a siRNA molecule, oractivates gene expression, such as an activating RNA molecule. Inanother embodiment, the active agent is a small molecule drug orpeptide. In one embodiment, the delivery moiety is a ligand for atoll-like receptor (such as oligonucleotides described herein). Inanother embodiment, the delivery moiety is another cell-specific ligand(such as aptamers).

In a second aspect, the present invention provides a method for thetreatment of diseases which comprises using the novel molecules of thepresent invention. Diseases which can be treated in accordance with thepresent invention include cancer, infectious diseases, autoimmunediseases, diseases due to excessive angiogenesis and diseases that canbenefit from increased angiogenesis. Cancers which can be treated withthe molecules of the present invention include, but are not limited to,melanoma, skin cancer, precancerous skin lesions, breast cancer,prostate cancer, lung cancer, glioma, pancreatic cancer, head and neckcancer, multiple myeloma, leukemias, lymphomas. Examples of infectiousdiseases include, but are not limited to, HIV, HPV infection andhepatitis. Examples of autoimmune diseases include, but are not limitedto, psoriasis, multiple sclerosis (MS) and inflammatory bowel disease(IBD). Examples of diseases due to excessive angiogenesis include, butare not limited to, cancer, diabetic retinopathy and Kaposi's Sarcoma.Examples of diseases that can benefit from increased angiogenesisinclude, but are not limited to, diseases needing wound repair(healing). The molecules of the present invention are administered topatients in need of treatment using conventional pharmaceuticalpractices.

In a third aspect, the present invention provides active agents that arecapable of acting in the Stat3 pathway which, when taken up by the cellsof interest, results in the treatment diseases including, but notlimited to cancer, infectious diseases and autoimmune diseases.

The molecules of the present invention have several advantages thatresult from the characteristics of the molecules. These advantagesinclude:

(a) ease of use and cost effectiveness primarily because of a reductionin the need to use transfection reagents;

(b) simplicity primarily because of the ability to make the molecules bychemical synthesis using standard synthesizers;

(c) versatility primarily because the molecules of the present inventioncan be easily adapted for various gene targets and modified be furthermodified for small molecule drug or peptide delivery with the use ofappropriate chemical linkers; and

(d) flexibility primarily because a similar design can be adapted fordifferent cell types capable of ODN or ORN uptake.

An “oligonucleotide” or “oligo” shall mean multiple nucleotides (i.e.molecules comprising a sugar (e.g. ribose or deoxyribose) linked to aphosphate group and to an exchangeable organic base, which is either asubstituted pyrimidine (e.g. cytosine (C), thymine (T) or uracil (U)) ora substituted purine (e.g. adenine (A) or guanine (G)). The term“oligonucleotide” as used herein refers to both oligoribonucleotides(ORNs) and oligodeoxyribonucleotides (ODNs). The term “oligonucleotide”shall also include oligonucleosides (i.e. an oligonucleotide minus thephosphate) and any other organic base containing polymer.Oligonucleotides can be obtained from existing nucleic acid sources(e.g. genomic or cDNA), but are preferably synthetic (e.g. produced byoligonucleotide synthesis).

A “stabilized oligonucleotide” shall mean an oligonucleotide that isrelatively resistant to in vivo degradation (e.g. via an exo- orendo-nuclease). Preferred stabilized oligonucleotides of the instantinvention have a modified phosphate backbone. Especially preferredoligonucleotides have a phosphorothioate modified phosphate backbone(i.e. at least one of the phosphate oxygens is replaced by sulfur).Other stabilized oligonucleotides include: nonionic DNA analogs, such asalkyl- and aryl-phosphonates (in which the charged phosphonate oxygen isreplaced by an alkyl or aryl group), phosphodiester andalkylphosphotriesters, in which the charged oxygen moiety is alkylated.Oligonucleotides which contain a diol, such as tetraethyleneglycol orhexaethyleneglycol, at either or both termini have also been shown to besubstantially resistant to nuclease degradation.

A “CpG containing oligonucleotide,” “CpG ODN” or “CpG ORN” refers to anoligonucleotide, which contains a cytosine/guanine dinucleotidesequence. Preferred CpG oligonucleotides are between 2 to 100 base pairsin size and contain a consensus mitogenic CpG motif represented by theformula:5′X₁X₂CGX₃X₄3′wherein C and G are unmethylated, X₁, X₂, X₃ and X₄ are nucleotides anda GCG trinucleotide sequence is not present at or near the 5′ and 3′ends. Examples of CpG ODNs are described in U.S. Pat. Nos. 6,194,388 and6,207,646, each incorporated herein by reference. Preferably the CpGoligonucleotides range between 8 and 40 base pairs in size. In addition,the CpG oligonucleotides are preferably stabilized oligonucleotides,particularly preferred are phosphorothioate stabilized oligonucleotides.The CpG ODNs or CpG ORNs can be synthesized as an oligonucleotide.Alternatively, CpG ODNs or CpG ORNs can be produced on a large scale inplasmids.

An “aptamer” refers to a nucleic acid molecule that is capable ofbinding to a particular molecule of interest with high affinity andspecificity (Tuerk and Gold, 1990; Ellington and Szostak, 1990). Thebinding of a ligand to an aptamer, which is typically RNA, changes theconformation of the aptamer and the nucleic acid within which theaptamer is located. The conformation change inhibits translation of anmRNA in which the aptamer is located, for example, or otherwiseinterferes with the normal activity of the nucleic acid. Aptamers mayalso be composed of DNA or may comprise non-natural nucleotides andnucleotide analogs. An aptamer will most typically have been obtained byin vitro selection for binding of a target molecule. However, in vivoselection of an aptamer is also possible. An aptamer will typically bebetween about 10 and about 300 nucleotides in length. More commonly, anaptamer will be between about 30 and about 100 nucleotides in length.See, e.g., U.S. Pat. No. 6,949,379, incorporated herein by reference.Examples of aptamers that are useful for the present invention include,but are not limited to, PSMA aptamer (McNamara et al., 2006), CTLA4aptamer (Santulli-Marotto et al., 2003) and 4-1BB aptamer (McNamara etal., 2007).

As used herein, the terms “Toll-like receptor” or “TLR” refer to anymember of a family of at least ten highly conserved mammalian patternrecognition receptor proteins (TLR1-TLR10) which recognizepathogen-associated molecular patterns (PAMPs) and act as key signalingelements in innate immunity. TLR polypeptides share a characteristicstructure that includes an extracellular (extracytoplasmic) domain thathas leucine-rich repeats, a transmembrane domain, and an intracellular(cytoplasmic) domain that is involved in TLR signaling. TLRs include,but are not limited, to human TLRs. TLRs include, but are not limited toTLR9, TLR8 and TLR3.

As used herein, the terms “TLR ligand” or “ligand for a TLR” refer to amolecule, that interacts, directly or indirectly, with a TLR through aTLR domain and is capable of being internalized by cells. In oneembodiment a TLR ligand is a natural ligand, i.e., a TLR ligand that isfound in nature. In one embodiment a TLR ligand refers to a moleculeother than a natural ligand of a TLR, e.g., a molecule prepared by humanactivity, such as a CpG containing oligonucleotide.

In accordance with the present invention, target cells for ODN- orORN-mediated delivery include any cell that is capable of internalizinga TLR ligand. Such cells include (a) cells of the myeloid lineageincluding dendritic cells, macrophages and monocytes, (b) cells of thelymphoid lineage including B cells and T cells, (c) endothelial cellsand (d) malignant cells being derivatives of the previously mentionedcells, e.g., multiple myeloma, B cell lymphoma and T cell lymphoma. Themalignant cells can also be any cells that possess the capacity ofuptaking and/or internalizing a TLR ligand.

In accordance with the present invention, novel molecules are providedby an active moiety for delivering an active agent to a cell of interestfor the treatment of diseases as disclosed herein. The novel moleculescomprises one or more of a first moiety that directs cell or tissuespecific delivery of the novel molecule linked to one or more of asecond moiety that is an active agent useful for treating cancer orother diseases. The moieties can be linked together directly or they canbe linked together indirectly through a linker. In one embodiment, thenovel molecule comprises two moieties as one molecule that ismultifunctional. For example, a TLR ligand and an siRNA are made intoone molecule for delivery, immune stimulation and blockingimmunosuppressive elements, such as Stat3, and/or oncogenic effects,such as caused by Stat3. In another embodiment, the novel moleculecomprises moieties attached to a linker that is multifunctional, suchthat it can contain a multitude of moieties. The linkage of the firstand second moieties can be provided through diverse structures and/orchemistry. The linkage can also be designed to allow for one firstmoiety to be linked to multiple second moieties. The linkage can bedesigned to allow for linkage of a first moiety to small molecule drugsor peptides.

In one embodiment, the molecule may have the structure A-X-B. In anotherembodiment, the molecule may have the structure

where X is a linker between the A and B moieties or between the A, B, Yand Z moieties. In one embodiment, we can make 2 or (n)-element chains,stars, branches (or mixtures thereof) etc and defining the chemistry andvalency of the linker(s). Valency can be substrate specific to controlpolymerization. In one embodiment, X may be multifunctional reactivemolecule having, e.g., NNP, where N is a nucleic acid binding sites andP is a peptide binding site. The linker may be derivatized, e.g., withFITC, such that the X moiety itself is also functional. In thisembodiment, X may be derivatized with a fluorochrome or similarmolecule, or may be derivatized with a chemotherapeutic agent.

In one embodiment, A, B, etc., i.e., any moiety attached to the linker,can be small molecules, peptides, polypeptides, proteins, antibodies andfragments thereof, other molecules such as lectins, DNA, RNA, ds RNA dsDNA, RNA/DNA hybrids (and modifications thereto), locked nucleic acids,RNA with 5′ triphosphates, antibodies, antibody fragments, antigens orantigen fragments.

In one embodiment, the function of A, B, etc., i.e., any moiety attachedto the linker, can be selected to include from delivery (includingapproaches to target to cells, tissues, organs), improvedpharmacokinetic properties, cytotoxic, cytostatic, apoptotic, genemodulating (including upregulation, e.g., activating RNA, ordownregulation, e.g., siRNA), pro-inflammatory, anti-inflammatory,antigenic, immunogenic pro-coagulant, anti-coagulant properties,pro-drug elements and combinations thereof. In another embodiment, eachof these moieties can modified as known in current state of art toimprove their desired properties. These (A, B or desired modifications)can also be selected for via screening, evolution or combinatorialapproaches as is well known to the skilled artisan.

In one embodiment, moieties that can be used for delivery include CpGODNs, CpG ORNs, polyG (Peng et al., 2005), poly(I:C) (Alexopoulou etal., 2001) (such as ligands for toll-like receptors (TLRs)) andaptamers. The TLR ligands are useful for delivering the molecules of thepresent invention to cells that are capable of internalizing TLRligands. Aptamers are useful for delivering the molecules of the presentinvention to cells which specifically bind the aptamers.

In one embodiment, some elements or moieties may be themselvesbifunctional or derivatized to be bifunctional or have improved function(e.g., adding a 5′ triphosphate on a CpG may be an enhanced stimulatorof intracellular and/or extracellular signaling).

The present invention also provides for linkers and/or methods forproviding the molecules of the present invention. In one embodiment, amolecule of the present invention is prepared by linking a first moiety,e.g. a CpG ODN, CpG ORN, oligonucleotides or aptamer, to a secondmoiety, e.g., a dsRNA, using multiple units of the C3 spacer as thelinker (Dela et al., 1987). A method for preparing such a molecule inwhich the first moiety is a CpG ODN is shown in the Examples.

In an embodiment in which the first moiety is an ODN, ORN,oligonucleotides or aptamer and the second moiety is a dsRNA, a moleculeof the present invention can be prepared by providing a dsRNA in whichone of the strands has an overhang and the first moiety has acomplementary overhang. The overhang can be spaced from the first moietyand the dsRNA by using linkers comprising multiple units of the C3spacer. After annealing, both components are connected creating adesired construct. By controlling the length of the overhang and itsmakeup we can control the strength and the specificity of theattachment. The preferred component of the overhang are: 2′-O-methyl RNA(2′-OMe), 2′-Fluoro RNA (2′-F) or Locked Nucleic Acids (LNAs) or PNA.Extremely high melting temperatures of an LNA/LNA duplex allow for theuse of much shorter overhangs. 2′-Fluoro RNA (2′-F) were reported tohave lower toxicity then 2′-O-methyl RNA (2′-OMe). Since the cost of LNAis still 10-15 times higher then 2′-Fluoro RNA (2′-F) the latter seemsto be the optimal choice for overhang component. Use of all of the aboveincreases the resistance of the oligonucleotide to cellular nucleases.See, for example, Kurreck et al. (2002, Braasch et al. (2002) andBraasch et al. (2003). The other exemplary sugar modifications include,for example, a 2′-O-methoxyethyl nucleotide, a 2′-O-NMA, a 2′-DMAEOE, a2′-AP, 2′-hydroxy, or a 2′-ara-fluoro or extended nucleic acid (ENA),hexose nucleic acid (HNA), or cyclohexene nucleic acid (CeNA). The useof overhangs for the construction allows for: (i) use of smallermolecules, (ii) higher purity at lower cost, (iii) lower cost of finalproduct and (iv) flexibility (construction of product on demand;possibility of matching of one component with multiple components). Theuse of a universal overhangs allows for the interchangeability of thecomponents.

The use of branching or bridging compounds allows for the synthesis of acomponent carrying two or more overhangs. Such branching or bridgingcompounds allows for the attachment of multiple first moiety components,e.g., CpG ODN, to the second moiety component, e.g., dsRNA, and/or forthe attachment of multiple second moiety components to the multiplefirst moiety components. The use of molecules having in multipleoverhangs allows for the assembly of complementary constructs consistingof two or more aptamers. Constructs of this kind would be used in thedimerization experiments. The use of molecules having multiple overhangsallows for the assembly of complementary constructs consisting of anaptamer and two or more siRNA duplexes.

Covalent constructs can also be prepared to form the molecules of thepresent invention. In this embodiment, the first and second moietieshave reactive groups. A covalent bond is created during the chemicalreaction between the reactive groups. Examples of such pairs of thereactive groups are as follows.

(A) carboxyl group and amino group. The attachment to be achieved bycreating a covalent bond between the carboxyl group on one component andthe amino group at the other component; it is possible to use acarbodimide to create the covalent bond.

(B) azide and acetylene groups. These groups combine readily with eachother—when held in close proximity—to form triazoles. Click chemistry isthe use of chemical building blocks with “built-in high-energy contentto drive a spontaneous and irreversible linkage reaction withappropriate complementary sites in other blocks,” Use of theazide-acetylene reaction represents “true progress” because of its highselectivity.

(C) vinyl sulfones and sulfuhydryl group, vinyl sulfones and terminalphosphothioesters, vinyl sulfones and amino group. Vinyl sulfones andsubstituted divinyl sulfones readily react with sulfuhydryl group (SH)in pH 5-7, with and terminal phosphothioesters in pH7, and with primaryand secondary amines at higher pH.

Conjugation of two biopolymers with the use of click chemistry (asdescribed above) can also be used to create the molecules of the presentinvention. Reaction of dsRNA component having multiple reactive groupswith the excess of the CPG or aptamer component leads to the productsconsisting of multiple dsRNAs attached to the single CPG or aptamercomponent. Reaction of first moiety having multiple reactive groups withthe excess of the small molecule drug leads to the products consistingof multiple drug molecules attached to a single CpG or aptamercomponent. Drugs may be attached to the constructs through thehydrolysable-digestible linker, such as a short peptide hydrolysable byesterase, to facilitate its release upon delivery to the target.

In one aspect, the active agents of the present invention are doublestranded RNA molecules. These double stranded RNA molecules may beuseful for downregulating gene expression, such as siRNA molecules.Alternatively, the double stranded RNA molecules may be useful forupregulating gene transcription, such as activating RNA molecules.

The siRNA molecule may have different forms, including a single strand,a paired double strand (dsRNA) or a hairpin (shRNA) and can be produced,for example, either synthetically or by expression in cells. In oneembodiment, DNA sequences for encoding the sense and antisense strandsof the siRNA molecule to be expressed directly in mammalian cells can beproduced by methods known in the art, including but not limited to,methods described in U.S. published application Nos. 2004/0171118,2005/0244858 and 2005/0277610, each incorporated herein by reference.The siRNA molecules are coupled to carrier molecules, such as CpGoligonucleotides, various TLR-ligands (such as polyG or poly(I:C) or RNAaptamers, using the techniques known in the art or described herein.

In one aspect, DNA sequences encoding a sense strand and an antisensestrand of a siRNA specific for a target sequence of a gene areintroduced into mammalian cells for expression. To target more than onesequence in the gene (such as different promoter region sequences and/orcoding region sequences), separate siRNA-encoding DNA sequences specificto each targeted gene sequence can be introduced simultaneously into thecell. In accordance with another embodiment, mammalian cells may beexposed to multiple siRNAs that target multiple sequences in the gene.

The siRNA molecules generally contain about 19 to about 30 base pairs,and may be designed to cause methylation of the targeted gene sequence.In one embodiment, the siRNA molecules contain about 19-23 base pairs,and preferably about 21 base pairs. In another embodiment, the siRNAmolecules contain about 24-28 base pairs, and preferably about 26 basepairs. In a further embodiment, the dsRNA has an asymmetric structure,with the sense strand having a 25-base pair length, and the antisensestrand having a 27-base pair length with a 2 base 3′-overhang. See, forexample, U.S. published application Nos. 2005/0244858, 2005/0277610 and2007/0265220, each incorporated herein by reference. In anotherembodiment, this dsRNA having an asymmetric structure further contains 2deoxynucleotides at the 3′ end of the sense strand in place of two ofthe ribonucleotides. Individual siRNA molecules also may be in the formof single strands, as well as paired double strands (“sense” and“antisense”) and may include secondary structure such as a hairpin loop.Individual siRNA molecules could also be delivered as precursormolecules, which are subsequently altered to give rise to activemolecules. Examples of siRNA molecules in the form of single strandsinclude a single stranded anti-sense siRNA against a non-transcribedregion of a DNA sequence (e.g. a promoter region).

The sense and antisense strands anneal under biological conditions, suchas the conditions found in the cytoplasm of a cell. In addition, aregion of one of the sequences, particularly of the antisense strand, ofthe dsRNA has a sequence length of at least 19 nucleotides, whereinthese nucleotides are adjacent to the 3′ end of antisense strand and aresufficiently complementary to a nucleotide sequence of the RNA producedfrom the target gene.

The RNAi molecule, may also have one or more of the following additionalproperties: (a) the antisense strand has a right shift from the typical21mer and (b) the strands may not be completely complementary, i.e., thestrands may contain simple mismatch pairings. A “typical” 21mer siRNA isdesigned using conventional techniques, such as described above. This21mer is then used to design a right shift to include 1-7 additionalnucleotides on the 5′ end of the 21mer. The sequence of these additionalnucleotides may have any sequence. Although the added ribonucleotidesmay be complementary to the target gene sequence, full complementaritybetween the target sequence and the siRNA is not required. That is, theresultant siRNA is sufficiently complementary with the target sequence.The first and second oligonucleotides are not required to be completelycomplementary. They only need to be substantially complementary toanneal under biological conditions and to provide a substrate for Dicerthat produces a siRNA sufficiently complementary to the target sequence.In one embodiment, the dsRNA has an asymmetric structure, with theantisense strand having a 25-base pair length, and the sense strandhaving a 27-base pair length with a 2 base 3′-overhang. In anotherembodiment, this dsRNA having an asymmetric structure further contains 2deoxynucleotides at the 3′ end of the antisense strand.

Suitable dsRNA compositions that contain two separate oligonucleotidescan be linked by a third structure. The third structure will not blockDicer activity on the dsRNA and will not interfere with the directeddestruction of the RNA transcribed from the target gene. In oneembodiment, the third structure may be a chemical linking group. Manysuitable chemical linking groups are known in the art and can be used.Alternatively, the third structure may be an oligonucleotide that linksthe two oligonucleotides of the dsRNA is a manner such that a hairpinstructure is produced upon annealing of the two oligonucleotides makingup the dsRNA composition. The hairpin structure will not block Diceractivity on the dsRNA and will not interfere with the directeddestruction of the RNA transcribed from the target gene.

The sense and antisense sequences may be attached by a loop sequence.The loop sequence may comprise any sequence or length that allowsexpression of a functional siRNA expression cassette in accordance withthe invention. In a preferred embodiment, the loop sequence containshigher amounts of uridines and guanines than other nucleotide bases. Thepreferred length of the loop sequence is about 4 to about 9 nucleotidebases, and most preferably about 8 or 9 nucleotide bases.

In another embodiment of the present invention, the dsRNA, i.e., theRNAi molecule, has several properties which enhances its processing byDicer. According to this embodiment, the dsRNA has a length sufficientsuch that it is processed by Dicer to produce an siRNA and at least oneof the following properties: (i) the dsRNA is asymmetric, e.g., has a 3′overhang on the sense strand and (ii) the dsRNA has a modified 3′ end onthe antisense strand to direct orientation of Dicer binding andprocessing of the dsRNA to an active siRNA. According to thisembodiment, the longest strand in the dsRNA comprises 24-30 nucleotides.In one embodiment, the sense strand comprises 24-30 nucleotides and theantisense strand comprises 22-28 nucleotides. Thus, the resulting dsRNAhas an overhang on the 3′ end of the sense strand. The overhang is 1-3nucleotides, such as 2 nucleotides. The antisense strand may also have a5′ phosphate.

Modifications can be included in the dsRNA, i.e., the RNAi molecule, solong as the modification does not prevent the dsRNA composition fromserving as a substrate for Dicer. In one embodiment, one or moremodifications are made that enhance Dicer processing of the dsRNA. In asecond embodiment, one or more modifications are made that result inmore effective RNAi generation. In a third embodiment, one or moremodifications are made that support a greater RNAi effect. In a fourthembodiment, one or more modifications are made that result in greaterpotency per each dsRNA molecule to be delivered to the cell.Modifications can be incorporated in the 3′-terminal region, the5′-terminal region, in both the 3′-terminal and 5′-terminal region or insome instances in various positions within the sequence. With therestrictions noted above in mind any number and combination ofmodifications can be incorporated into the dsRNA. Where multiplemodifications are present, they may be the same or different.Modifications to bases, sugar moieties, the phosphate backbone, andtheir combinations are contemplated. Either 5′-terminus can bephosphorylated.

In another embodiment, the antisense strand is modified for Dicerprocessing by suitable modifiers located at the 3′ end of the antisensestrand, i.e., the dsRNA is designed to direct orientation of Dicerbinding and processing. Suitable modifiers include nucleotides such asdeoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and thelike and sterically hindered molecules, such as fluorescent moleculesand the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl groupfor the 2′-deoxyribofuranosyl sugar normally present in dNMPs. Othernucleotide modifiers could include 3′-deoxyadenosine (cordycepin),3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI),2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxynucleotides are used as the modifiers. When nucleotide modifiersare utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers aresubstituted for the ribonucleotides on the 3′ end of the antisensestrand. When sterically hindered molecules are utilized, they areattached to the ribonucleotide at the 3′ end of the antisense strand.Thus, the length of the strand does not change with the incorporation ofthe modifiers. In another embodiment, the invention contemplatessubstituting two DNA bases in the dsRNA to direct the orientation ofDicer processing. In a further invention, two terminal DNA bases arelocated on the 3′ end of the antisense strand in place of tworibonucleotides forming a blunt end of the duplex on the 5′ end of thesense strand and the 3′ end of the antisense strand, and atwo-nucleotide RNA overhang is located on the 3′-end of the sensestrand. This is an asymmetric composition with DNA on the blunt end andRNA bases on the overhanging end.

Examples of modifications contemplated for the phosphate backboneinclude phosphonates, including methylphosphonate, phosphorothioate, andphosphotriester modifications such as alkylphosphotriesters, and thelike. Examples of modifications contemplated for the sugar moietyinclude 2′-alkyl pyrimidine, such as 2′-O-methyl, 2′-fluoro, amino, anddeoxy modifications and the like (see, e.g., Amarzguioui et al., 2003).Examples of modifications contemplated for the base groups includeabasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil,5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil and the like.Locked nucleic acids, or LNA's, could also be incorporated. Many othermodifications are known and can be used so long as the above criteriaare satisfied. Examples of modifications are also disclosed in U.S. Pat.Nos. 5,684,143, 5,858,988, 6,291,438 and 7,307,069 and in U.S. publishedpatent application No. 2004/0203145, each incorporated herein byreference. Other modifications are disclosed in Herdewijn (2000),Eckstein (2000), Rusckowski et al. (2000), Stein et al. (2001) andVorobjev et al. (2001), each incorporated herein by reference.

Additionally, the siRNA structure can be optimized to ensure that theoligonucleotide segment generated from Dicer's cleavage will be theportion of the oligonucleotide that is most effective in inhibiting geneexpression. For example, in one embodiment of the invention a 27-bpoligonucleotide of the dsRNA structure is synthesized wherein theanticipated 21 to 22-bp segment that will inhibit gene expression islocated on the 3′-end of the antisense strand. The remaining baseslocated on the 5′-end of the antisense strand will be cleaved by Dicerand will be discarded. This cleaved portion can be homologous (i.e.,based on the sequence of the target sequence) or non-homologous andadded to extend the nucleic acid strand.

Activating RNA molecules are similar in design as siRNA molecules.However, they can also be shorter than siRNA molecules. Thus, activatingRNA molecules may be 12-30 nucleotides in length, although a length of18-30 nucleotides is preferred. Activating RNA molecules are targeted tothe promoter region of the gene of interest and are designed to inducetranscriptional activation. In one embodiment, the region within thepromoter of the gene is selected from a partially single-strandedstructure, a non-B-DNA structure, an AT-rich sequence, a cruciform loop,a G-quadruplex, a nuclease hypersensitive elements (NHE), and a regionlocated between nucleotides −100 to +25 relative to a transcriptionstart site of the gene. See, for example, Li et al. (2006), Kuwabara etal. (2005), Janowski et al. (2007) and U.S. published application No.2007/0111963, each incorporated herein by reference. A broad spectrum ofchemical modifications can be made to duplex RNA, without negativelyimpacting the ability of the dsRNA to selectively increase synthesis ofthe target transcript. These chemical modifications included thosedescribed above for siRNA molecules as well as those described in U.S.published application No. 2007/0111963.

RNA for the siRNA or activating RNA component of the present inventionmay be produced enzymatically or by partial/total organic synthesis, andmodified ribonucleotides can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, each strand is preparedchemically. Methods of synthesizing RNA molecules are known in the art,in particular, the chemical synthesis methods as described in Verma andEckstein (1998).

In another aspect, the active agents of the present invention are smallmolecule drugs or peptides. Examples of small molecule drugs include,but are not limited to, Stat3 inhibitors (such as those commerciallyavailable from Calbiochem), Imatinib (Bcr-Abl), Sunitib (VEGF receptor),Sorefenib (Raf) and DASATINIB (Src). Examples of peptides include, butare not limited to, Stat3 peptidomimetics, p53 peptidomimetics andFarnesyl Transferase inhibitors.

The present invention further provides active agents that are capable ofacting in the Stat3 signaling pathway or affecting genes regulated byStat3. These active agents, when taken up by the cells of interest,result in the treatment of cancer or other diseases. In one embodiment,the active agent is an siRNA molecule directed against Stat3 and resultsin the down regulation of Stat3. In another embodiment, the active agentis an siRNA molecule directed against SOCS3 which is an inhibitor ofStat3. In a further embodiment, the active agent is an activating RNAfor tumor suppressor genes.

In addition, the present invention provides a method for treatingdiseases. The molecules of the present invention are administered topatients in need of treatment using conventional pharmaceuticalpractices. Suitable pharmaceutical practices are described in Remington:The Science and Practice of Pharmacy, 21^(st) Ed., University ofSciences in Philadelphia, Ed., Philadelphia, 2005. In one embodiment,the present invention provides for the delivery of dsRNA, such as siRNAor activating RNA, for the treatment of cancer. In another embodiment,the present invention provides for the delivery of dsRNA for thetreatment of infectious diseases. In a further embodiment, the presentinvention provides the delivery of dsRNA for the treatment of autoimmunediseases. The dsRNA can be specifically delivered to cells as describedherein.

The present invention can also be used to deliver DNA or RNA that encodeantigens to cells, e.g., DCs to stimulate an immune response, e.g.,vaccine or immunomodulator. Suitable antigens could be tumor orinfectious agents, including but not limited to, virus, fungus,bacteria, rikettsia, amoeba.

Thus, the present invention relates to the use of multifunctionalmolecules to modulate cancer and the immune system. The presentinvention relates delivery of RNA (siRNA and/or activating RNA) by TLRligands as single molecule in vivo. The present invention is illustratedherein by a covalently linked siRNA and CpG molecule. In particular, weshow the (mouse) CpG motif coupled to a 27mer siRNA against Stat-3.Other TLR ligands, including but not limited to polyI:C, polyG LPS, andpeptidoglycan can also been linked to siRNAs for various target genes.

Stat3 is a ‘master switch’—in both cancer and tumor cells andtumor-associated immune cells—that controls tumor survival,angiogenesis/metastasis and immune evasion. The challenge is to turnStat3 off in the desired cells in cancer in patients. The presentinvention describes the development of optimal Stat3 siRNAs (Dicer) withantitumor effects in vivo, and shows that Stat3siRNA linked to CpGoligonucleotide efficiently enters dendritic cells. Targeting Stat3drastically improves CpG-based cancer. The utility of the presentinvention has been demonstrated herein using melanoma as the model.However, it is understood that the present invention is not limited tomelanoma but is equally applicable to all types of cancer.

Many promising immunotherapeutic approaches are in clinical trials formelanoma patients. However, these approaches face a major challenge:tumor-induced immune suppression. Since Stat3 is a key mediator oftumor-induced immunosuppression in melanoma, we reasoned that targetingStat3—although not perfect with current drugs—will significantly improvethe tumor immunologic microenvironment and thus enhance variousimmunotherapeutic approaches. As demonstrated herein, targeting Stat3dramatically improves CpG ODN-based melanoma immunotherapy. We show thatinhibiting Stat3 in myeloid cells, in conjunction with local CpGtreatment, can eliminate large (1.5 cm in diameter) established B16melanomas. We also demonstrate that targeting Stat3 systemically with asmall-molecule Stat3 inhibitor not only dramatically improve theantitumor effects at primary tumor sites receiving CpG injection butalso leads to concomitant antitumor effects on distal tumors without CpGtreatment. In addition to the potent antitumor effects, our resultsindicate that blocking Stat3 in tumor-stromal immune cells activatesStat1 and NF-κB, leading to Th-1 immune responses of diverse immunesubsets that are fundamental for numerous cancer immunotherapies.Consistent with the idea that targeting Stat3 can improveimmunotherapeutic efficacies are the findings by Kirkwood and colleagues(Kirkwood et al., 1999), who demonstrated that high dose IFNα-basedimmunotherapy response inversely correlates with Stat3 activity inmelanoma patients.

In another aspect, the present invention provides for a pharmaceuticalcomposition comprising of molecules of the present invention, i.e., themolecules that contain a cell specific delivery moiety and one or moreadditional active agents. The cell specific delivery moiety and theadditional active agent(s) may be directly linked together or they maybe indirectly linked together through the use of a linker. As describedherein, the active agent may be an siRNA, an activating RNA, a smallmolecule drug or a peptide. These molecules can be suitably formulatedand introduced into the environment of the cell by any means that allowsfor a sufficient portion of the sample to enter the cell to induce genesilencing, if it is to occur. Many formulations for dsRNA are known inthe art and can be used for delivery of the molecules of the presentinvention to mammalian cells so long as active agent gains entry to thetarget cells so that it can act. See, e.g., U.S. published patentapplication Nos. 2004/0203145 A1 and 2005/0054598 A1, each incorporatedherein by reference. For example, siRNA can be formulated in buffersolutions such as phosphate buffered saline solutions, liposomes,micellar structures, and capsids. Formulations of siRNA with cationiclipids can be used to facilitate transfection of the dsRNA into cells.For example, cationic lipids, such as lipofectin (U.S. Pat. No.5,705,188, incorporated herein by reference), cationic glycerolderivatives, and polycationic molecules, such as polylysine (publishedPCT International Application WO 97/30731, incorporated herein byreference), can be used. Suitable lipids include Oligofectamine,Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals,Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be usedaccording to the manufacturer's instructions.

It can be appreciated that the method of introducing the molecules ofthe present invention into the environment of the cell will depend onthe type of cell and the make up of its environment. For example, whenthe cells are found within a liquid, one preferable formulation is witha lipid formulation such as in lipofectamine and the molecules of thepresent invention can be added directly to the liquid environment of thecells. Lipid formulations can also be administered to animals such as byintravenous, intramuscular, or intraperitoneal injection, or orally orby inhalation or other methods as are known in the art. When theformulation is suitable for administration into animals such as mammalsand more specifically humans, the formulation is also pharmaceuticallyacceptable. Pharmaceutically acceptable formulations for administeringoligonucleotides are known and can be used. In some instances, it may bepreferable to formulate molecules of the present invention in a bufferor saline solution and directly inject the formulated dsRNA into cells,as in studies with oocytes. The direct injection of dsRNA duplexes mayalso be done. For suitable methods of introducing siRNA see U.S.published patent application No. 2004/0203145 A1, incorporated herein byreference.

Suitable amounts of molecules of the present invention must beintroduced and these amounts can be empirically determined usingstandard methods. Typically, effective concentrations of individualdsRNA species in the environment of a cell will be about 50 nanomolar orless 10 nanomolar or less, or compositions in which concentrations ofabout 1 nanomolar or less can be used. In other embodiment, methodsutilize a concentration of about 200 picomolar or less and even aconcentration of about 50 picomolar or less can be used in manycircumstances. Typically, effective doses of small molecule drugs orpeptides can be lower than previously used in view of the cell specificdelivery provided by the present invention.

The method can be carried out by addition of the compositions containingthe molecules of the present invention to any extracellular matrix inwhich cells can live provided that the composition is formulated so thata sufficient amount of the active agent can enter the cell to exert itseffect. For example, the method is amenable for use with cells presentin a liquid such as a liquid culture or cell growth media, in tissueexplants, or in whole organisms, including animals, such as mammals andespecially humans.

Expression of a target gene can be determined by any suitable method nowknown in the art or that is later developed. It can be appreciated thatthe method used to measure the expression of a target gene will dependupon the nature of the target gene. For example, when the target geneencodes a protein the term “expression” can refer to a protein ortranscript derived from the gene. In such instances the expression of atarget gene can be determined by measuring the amount of mRNAcorresponding to the target gene or by measuring the amount of thatprotein. Protein can be measured in protein assays such as by stainingor immunoblotting or, if the protein catalyzes a reaction that can bemeasured, by measuring reaction rates. All such methods are known in theart and can be used. Where the gene product is an RNA species expressioncan be measured by determining the amount of RNA corresponding to thegene product. The measurements can be made on cells, cell extracts,tissues, tissue extracts or any other suitable source material.

The determination of whether the expression of a target gene has beenreduced can be by any suitable method that can reliably detect changesin gene expression. Typically, the determination is made by introducinginto the environment of a cell undigested siRNA such that at least aportion of that siRNA enters the cytoplasm and then measuring theexpression of the target gene. The same measurement is made on identicaluntreated cells and the results obtained from each measurement arecompared. Similarly the determination can be made by introducing intothe environment of a cell undigested activating RNA such that at least aportion of that activating RNA enters the cytoplasm and then measuringthe expression of the target gene.

The molecules of the present invention can be formulated as apharmaceutical composition which comprises a pharmacologically effectiveamount of the molecules and pharmaceutically acceptable carrier. Apharmacologically or therapeutically effective amount refers to thatamount of a molecule of the present invention effective to produce theintended pharmacological, therapeutic or preventive result. The phrases“pharmacologically effective amount” and “therapeutically effectiveamount” or simply “effective amount” refer to that amount of a dsRNA,small molecule drug or peptide effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 20% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a20% reduction in that parameter.

The phrase “pharmaceutically acceptable carrier” refers to a carrier forthe administration of a therapeutic agent. Exemplary carriers includesaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract. The pharmaceutically acceptable carrier of thedisclosed dsRNA composition may be micellar structures, such as aliposomes, capsids, capsoids, polymeric nanocapsules, or polymericmicrocapsules.

Polymeric nanocapsules or microcapsules facilitate transport and releaseof the encapsulated or bound dsRNA into the cell. They include polymericand monomeric materials, especially including polybutylcyanoacrylate. Asummary of materials and fabrication methods has been published (seeKreuter, 1991). The polymeric materials which are formed from monomericand/or oligomeric precursors in the polymerization/nanoparticlegeneration step, are per se known from the prior art, as are themolecular weights and molecular weight distribution of the polymericmaterial which a person skilled in the field of manufacturingnanoparticles may suitably select in accordance with the usual skill.

Suitably formulated pharmaceutical compositions of this invention can beadministered by any means known in the art such as by parenteral routes,including intravenous, intramuscular, intraperitoneal, subcutaneous,transdermal, airway (aerosol), rectal, vaginal and topical (includingbuccal and sublingual) administration. In some embodiments, thepharmaceutical compositions are administered by intravenous orintraparenteral infusion or injection.

In general a suitable dosage unit of active agent moiety of themolecules of the present invention will be in the range of 0.001 to 0.25milligrams per kilogram body weight of the recipient per day, or in therange of 0.01 to 20 micrograms per kilogram body weight per day, or inthe range of 0.01 to 10 micrograms per kilogram body weight per day, orin the range of 0.10 to 5 micrograms per kilogram body weight per day,or in the range of 0.1 to 2.5 micrograms per kilogram body weight perday. Pharmaceutical composition comprising the siRNA can be administeredonce daily. However, the therapeutic agent may also be dosed in dosageunits containing two, three, four, five, six or more sub-dosesadministered at appropriate intervals throughout the day. In that case,the active agent, e.g., dsRNA, contained in each sub-dose must becorrespondingly smaller in order to achieve the total daily dosage unit.The dosage unit can also be compounded for a single dose over severaldays, e.g., using a conventional sustained release formulation whichprovides sustained and consistent release of the active agent, e.g.,dsRNA, over a several day period. Sustained release formulations arewell known in the art. In this embodiment, the dosage unit contains acorresponding multiple of the daily dose. Regardless of the formulation,the pharmaceutical composition must contain active agent, e.g., dsRNA,in a quantity sufficient to inhibit expression of the target gene in theanimal or human being treated. The composition can be compounded in sucha way that the sum of the multiple units of active agent togethercontain a sufficient dose.

Data can be obtained from cell culture assays and animal studies toformulate a suitable dosage range for humans. The dosage of compositionsof the invention lies within a range of circulating concentrations thatinclude the ED₅₀ (as determined by known methods) with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound that includes the IC₅₀ (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsof dsRNA in plasma may be measured by standard methods, for example, byhigh performance liquid chromatography.

In a further aspect, the present invention relates to a method for TGSin a mammalian, including human, cell. The method comprises introducingthe siRNA containing molecules of the present invention into theappropriate cell. The term “introducing” encompasses a variety ofmethods of introducing the siRNA containing molecules into a cell,either in vitro or in vivo, such as described above.

In a further aspect, the present invention relates to a method for geneactivation in a mammalian cell, including human cell. The methodcomprises introducing the activating RNA containing molecules of thepresent invention into the appropriate cell. The term “introducing”encompasses a variety of methods of introducing the siRNA containingmolecules into a cell, either in vitro or in vivo, such as describedabove.

In a further aspect, the present invention relates to a method fortreating a disease or physiological disorder or condition in a mammal,including a human. The method comprises introducing the small moleculedrug or peptide containing molecules of the present invention into theappropriate cell. The term “introducing” encompasses a variety ofmethods of introducing the siRNA containing molecules into a cell,either in vitro or in vivo, such as described above.

TLR ligands, such as CpG, are known to stimulate innate immunity. Thepresent invention illustrates that blocking Stat3, either genetically,or pharmacologically, results in drastically improved immune responsesand antitumor effects.

A major challenge facing siRNA-based therapies is efficient uptake ofsiRNA by desired cells in vivo. The present studies demonstrate that aTLR ligand, e.g., a moiety consisting of oligonucleotides that canactivate immune responses against cancer and infectious diseases when itis linked to siRNA, is able to mediate siRNA uptake and internalizationby desired immune cells. They include myeloid cells, such as macrophagesand dendritic cells, in cultured cells, and in animals through eitherintratumoral or intravenous injections of the chimeric constructs. Thisuptake occurs in the absence of any transfection agents. The DNA-RNAchimeric constructs can be processed by Dicer and is associated withDicer in living cells. In vivo delivery of the chimeric constructsresults in gene silencing in DCs and macrophages, including those residein tumors and the tumor draining lymph nodes. Similar constructinvolving TLR ligand and siRNA can also be uptaken by human monocytes,leading to gene silencing.

In addition to macrophages, dendritic cells and monocytes, the presentstudies show that the CpG-siRNA chimeric constructs can be efficientlytaken up by both human and mouse B cell malignant cells (B cell lymphomaand multiple myeloma).

Stat3 is a potent oncogenic transcriptional factor that is continuouslyactivated in diverse human cancer (Yu and Jove, 2004). Activated Stat3not only promotes tumor cell survival, proliferation and angiogenesis(Yu and Jove, 2004), it also mediates tumor immune suppression throughits activation in both tumor cells and in immune cells in the tumormicroenvironment (Wang et al., 2004; Kortylewski et al., 2005b; Yu etal., 2007). Effective targeting of Stat3 in tumor cells has been shownto induce tumor cell apoptosis, inhibit tumor cell proliferation,angiogenesis/metastasis (Yu and Jove, 2004). Inhibiting Stat3 in bothtumor cells and/or immune cells also elicits multi-component antitumorimmune responses (Wang et al., 2004; Kortylewski et al., 2005b).Although CpG is a potent immune stimulator, its effects in tumor-bearinghosts are dampened by the tumor microenvironment, which is, at least inpart, mediated by Stat3 activation. Interestingly, CpG, like severalother pathogen-associated immune stimulators, such as LPS, is anactivator of Stat3 (through activating IL-10, which in turn activatesStat3), and Stat3 serves as feedback mechanism to limit theirimmunostimulatory effects (Benkhart et al., 2000; Samarasinghe et al.,2006). These findings suggest that triggering toll-like receptor throughits ligand while blocking Stat3 should negate the inhibitory effectsassociated with CpG, thereby generating potent immune responses andimproving CpG treatment for both cancer and infectious diseases. Ourdata generated with CpG treatment in conjunction with genetic knockoutof Stat3 in myeloid cells prove this point. These data illustrate thatblocking Stat3, by any means, are highly desirable for enhancing theefficacies of TLR ligand-based therapies. See data and Examples herein.See also U.S. Patent Application Publication No. 2008/02144356, PCTInternational Publication No. WO 2008/094254, and U.S. PatentApplication Publication No. 2011/0071210, each incorporated herein inits entirety for all that it discloses.

As an example of the TLR ligand-siRNA chimeric construct, siRNA againstStat3 (SEQ ID NO:3 for sense strand; SEQ ID NO:2 for antisense strand)is linked to toll-like receptor 9 ligand, CpG oligonucleotide (ODN) (SEQID NO:1) (FIG. 2 a, top). Optimal sequences of both human and mouseStat3 siRNA have been selected (FIG. 7), followed by linkage to CpGsingle stranded ODN (FIG. 2 a, top), and other toll-like receptorligands (FIG. 8). The construct can be processed by Dicer (FIG. 2 a,lower), and is associated with Dicer in living cells (FIG. 2 d), andcauses gene silencing (FIGS. 2 e, 2 f). The chimeric constructs, whendelivered in vivo in tumor bearing mice, are efficiently uptaken andinternalized by targeted cells, such as macrophages and dendritic cells(FIG. 3 a). These immune cells are able to traffic from tumor to tumortraining lymph nodes, where they can interact with T cells (FIG. 3 a).In vivo gene silencing is also detected in dendritic cells andmacrophages in tumor draining lymph nodes (FIG. 3 b). The immunemodulation induced by the toll-like receptor 9 ligand-Stat3 siRNA leadsto potent antitumor effects on well established B16 melanoma (FIGS. 3c-3 e). Both local intratumoral injection and systemic intravenousinjection routes are tested, demonstrating the usefulness of theODN-siRNAs as therapeutic agents (FIGS. 3 c-3 e). CpG alone, Stat3siRNAalone, or CpG-linked to a scrambled siRNA are not able to inducesignificant antitumor effects, testifying the superior efficacies oflinking two active moieties: TLR9 ligand and Stat3 siRNA (FIG. 3 c-e).Tumor bearing mice treated with the CpG-Stat3siRNA constructs displayactivation of dendritic cells (FIG. 3 f), increased CD8+ T cells, NKcells and reduced number of T regulatory cells in the tumor and/or tumordraining lymph nodes (FIG. 3 g). Treating tumor-bearing mice withCpG-Stat3siRNA also increases tumor infiltrating tumor antigen-specificCD8+ T cells (FIG. 3 h).

Similarly, TLR ligand-siRNA chimeric constructs can also be taken up byhuman monocytes, leading to gene silencing (FIG. 4).

We have further shown that the CpG-Stat3siRNA is easily uptaken by bothmurine and human B malignant cells, including both lymphoma and multiplemyeloma cells (FIG. 5), many of which also express TLR (Bourke et al.,2003; Reid et al., 2005; Jahrsdorfer et al., 2005). We show that uptakeand internalization of the CpG-Stat3siRNA leads to gene silencing ofStat3 (FIG. 5 d), which is accompanied by increased cell cycle arrest ofthe myeloma cells relative to those treated with CpG-scrambled siRNA incell culture (FIG. 5 e). Furthermore, in vivo treatment with theCpG-Stat3siRNA construct leads to significant growth inhibition ofwell-established murine myeloma tumors (FIG. 6 a). Tumor growthinhibition due to CpG-Stat3siRNA in vivo treatment is associated withupregulation of co-stimulatory molecules on tumor-infiltrating dendriticcells (FIGS. 6 b, c).

The DNA (or RNA)-RNA constructs are synthesized chemically withoutinvolvement of enzymes. The success of CpG-Stat3siRNA chimeric moleculefor inducing immune responses and antitumor effects, through blockingStat3 in immune cells and/or in tumor cells, demonstrates a novelgeneral approach: using TLR ligand oligonucleotides, which include CpG,polyI:C (TLR3 ligand), polyG (TLR8 ligand), to deliver short RNA, whichinclude both siRNA and activating RNA, to desired cells in vitro and invivo, to stimulate innate immunity, to negate undesired effects and/orelicit desired effects through siRNA and/or activating RNA.

As a result, creating chimeric molecule consisting of TLR ligand andsiRNA and/or activating RNA, has great versatility and can be easilyadapted for various gene targets. It also has flexibility: similardesign can be adapted for different cell types capable of ODN/ORN uptakeand internalization. Using a linker, modification of such approach toinclude multiple active moieties, such as multiple siRNA, with TLRligand as a single agent for treating cancer and infectious disease isfeasible. This approach can also be modified to enable small moleculedrug delivery.

More specifically, the present invention relates to specific chimericmolecules that are useful for the treatment of diseases. Thus, in oneaspect, the present invention provides a standard conjugate for use inpreclinical and phase I studies. In accordance with this aspect, thechimeric molecule comprises the components:

A. Human STAT3 SS siRNA (sense strand; underlined aredeoxyribonucleotides):

(SEQ ID NO: 4) GGA AGC UGC AGA AAG AUA CGA CUG A;B. Human CpG(D19)-STAT3 AS siRNA (antisense strand; asterisks indicatephosphorothioated sites, X indicates single C3 carbon chainlinker/propanediol linker):

G*GT GCA TCG ATG CAG G*G*G* G*G XXXXX UCA GUC GUA UCU UUC UGC AGC UUC CGU. (SEQ ID NO: 16 XXXXX SEQ ID NO: 5)

In a second aspect, the present invention provides a chimeric moleculethat is prepared to include modification sites for the sense strandsequence to produce a conjugate with increased serum stability. Inaccordance with this aspect, the modified sense strand comprises:

A. Human STAT3 SS siRNA (sense strand; underlined aredeoxyribonucleotides; bold are chemically modified for increasednuclease resistance, e.g. 2′F-, 2′OMe-, LNA, nucleotides or othermodifications described herein):

(SEQ ID NO: 17) GGA AGC UGC AGA AAG AUA CGA CU G A.In one embodiment, this sense strand is combined with the humanCpG(D19)-STAT3 AS siRNA described above.

In a third aspect, the present invention provides an alternativethree-component conjugate with complementary “sticky ends” instead offixed propanediol linker between CpG and siRNA moieties (to simplifysynthesis). In accordance with this aspect, the chimeric moleculecomprises the components:

A. Human STAT3 SS siRNA-overhang (sense strand; X indicates single C3carbon chain linker/propanediol linker; bold are chemically modified2′F- or 2′OMe-nucleotides or other modifications as described herein):

GGA AGC UGC AGA AAG AUA CGA CUG A XXXXX ACG UGG  CCG ACU UCC U;(SEQ ID NO: 18 XXXXX SEQ ID NO: 19)B. Human CpG(D19)-overhang (asterisks indicate phosphorothioated sites;X indicates single C3 carbon chain linker/propanediol linker; bold arechemically modified 2′F- or 2′OMe-nucleotides or other modifications asdescribed herein):

G*GT GCA TCG ATG CAG G*G*G* G*G XXXXX AGG AAG UCG GCC ACG U;(SEQ ID NO: 5 XXXXX SEQ ID NO: 20)C. Human STAT3 AS siRNA (antisense strand):

(SEQ ID NO: 5) UCA GUC GUA UCU UUC UGC AGC UUC CGU.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.);Ausubel et al., 1992), Current Protocols in Molecular Biology (JohnWiley & Sons, including periodic updates); Glover, 1985, DNA Cloning(IRL Press, Oxford); Russell, 1984, Molecular biology of plants: alaboratory course manual (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Anand, Techniques for the Analysis of ComplexGenomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide toYeast Genetics and Molecular Biology (Academic Press, New York, 1991);Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.); Nucleic Acid Hybridization (B. D. Hames & S.J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S.J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6thEdition, Blackwell Scientific Publications, Oxford, 1988; Fire et al.,RNA Interference Technology: From Basic Science to Drug Development,Cambridge University Press, Cambridge, 2005; Schepers, RNA Interferencein Practice, Wiley-VCH, 2005; Engelke, RNA Interference (RNAi): The Nuts& Bolts of siRNA Technology, DNA Press, 2003; Gott, RNA Interference,Editing, and Modification: Methods and Protocols (Methods in MolecularBiology), Human Press, Totowa, N.J., 2004; Sohail, Gene Silencing by RNAInterference: Technology and Application, CRC, 2004.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1 Materials and Methods for Examples 1-10

Cells Lines:

Mouse B16 melanoma cells were purchased from American Type CultureCollection. Human peripheral blood mononuclear cells (PBMC) from healthydonors were collected by apheresis and mononuclear cells were isolatedover a Ficoll gradient.

Oligonucleotide Design and Synthesis:

The phosphothioated oligodeoxynucleotide (ODN) and antisense strands(AS) of siRNAs were linked using 6 units of C3 spacer (Glen Research,San Diego, Calif.). The resulting constructs were hybridized withcomplementary sense strands (SS) of siRNAs to create chimeric ODN-siRNAconstructs used in the study (deoxynucleotides are shown underlined).Sequences of single stranded constructs are listed below. See also FIG.8.

Mouse Stat3 siRNA (SS) (SEQ ID NO: 3) 5′ CAGGGUGUCAGAUCACAUGGGCUAA 3′CpG1668-mouse Stat3 siRNA(AS) 5′TCCATGACGTTCCTGATGCT-linker-UUAGCCCAUGUGAUCUGACACCCUGAA 3′(SEQ ID NO: 1-linker-SEQ ID NO: 2) GpC-mouse Stat3 siRNA (AS) 5′TCCATGAGCTTCCTGATGCT-linker-UUAGCCCAUGUGAUCUGACACCCUGAA 3′(SEQ ID NO: 6-linker-SEQ ID NO: 2) Human STAT3 siRNA (SS) (SEQ ID NO: 4)5′ GGAAGCUGCAGAAAGAUACGACUGA 3′ CpG(D19)-human STAT3 siRNA (AS) 5′GGTGCATCGATGCAGGGGGG-linker-UCAGUCGUAUCUUUCUGCAGCUUCCGU 3′(SEQ ID NO: 1-linker-SEQ ID NO: 5) Scrambled RNA (SS) (SEQ ID NO: 8 5′UCCAAGUAGAUUCGACGGCGAAGTG 3′ CpG1668-scrambled RNA (AS) 5′TCCATGACGTTCCTGATGCT-linker-CACUUCGCCGUCGAAUCUACUUGGAUU 3′(SEQ ID NO: 1-linker-SEQ ID NO: 9

The correct formation of siRNA duplex was confirmed by in vitro Dicercleavage assays. 0.5 μg of each ODN-siRNA construct was subjected toprocessing by 1 U of Dicer (Ambion) in 37° C. for 1.5 hr, resolved with15% polyacrylamide/7.5M urea gel and results of the dicing reaction werevisualized with SYBR Gold staining (Invitrogen).

Quantitative Real-Time PCR:

Total RNA was extracted from cultured or primary cells using RNeasy kit(Qiagen). After cDNA synthesis using iScript cDNA Synthesis kit(Bio-Rad), samples were analyzed using pairs of primers specific forStat3, GAPDH mRNAs or 18S rRNA (SuperArray Bioscience Corporation).Sequence-specific amplification was detected by fluorescent signal ofSYBR Green (Bio-Rad) by using Chromo4 Real-time PCR Detector (Bio-Rad).

Electromobility Shift Assay (EMSA) and Western Blot:

EMSA and western blot analyses to detect Stat3 DNA-binding and proteinexpression were performed as described previously (Wang et al., 2004).

In Vivo Experiments:

Mouse care and experimental procedures were performed underpathogen-free conditions in accordance with established institutionalguidance and approved protocols from Research Animal Care Committees ofthe City of Hope. We obtained Mx1-Cre mice from the Jackson Laboratoryand Stat3^(flox/flox) mice from S. Akira and K. Takeda. Generation ofmice with Stat3^(−/−) hematopoietic cells by inducible Mx1-Crerecombinase system has been reported (Kortylewski et al., 2005b; Lee etal., 2002). For s.c. tumor challenge, we injected 1×10⁵ B16 tumor cellsinto 7-8 weeks old transgenic mice 4 d after poly(I:C)-treatment toinduce Stat3 ablation. After tumors reached average size of ca. 1 cm,mice were injected peritumorally with 0.78 nmole of phosphothioated CpGODN (CpG1668; SEQ ID NO:1) or control GpG ODN (GpC; SEQ ID NO:6)) insome experiments, and tumor growth was monitored three times a week. Forthe analysis of cellular and molecular mechanisms of CpG ODN effect,mice were sacrificed at 1, 2 or 3 d post-CpG treatment, and spleens,lymph nodes as well as tumor specimens were harvested. For celldepletion experiments in vivo, mice were pretreated with anti-CD8 andanti-CD4 antibodies (clone 2.43 and GK1.5, respectively) oranti-asialo-GM1 serum (Wako) before tumor inoculation and then wereinjected at weekly intervals during the course of the experiment. Inexperiments on CpG-mediated siRNA delivery, mice were challenged withB16 tumors 6 days or 2 days before starting intratumoral or intravenousinjections of 0.78 nmole ODN-RNA fusion constructs, respectively.Treatment continued every second day for 2-3 weeks, mice were sacrificedand immune effects of treatments were analyzed.

Flow Cytometry and ELISA:

We prepared single cell suspensions of spleen, lymph node or tumortissues by mechanic dispersion followed by collagenase D/DNase Itreatment as described before (Kortylewski et al., 2005b). Forextracellular staining of mouse immune markers 1×10⁶ of freshly preparedcells suspended in PBS/2% FCS/0.1% w/v sodium azide was preincubatedwith FcγIII/IIR-specific antibody to block non-specific binding andstained with different combinations of fluorochrome-coupled antibodiesto CD11c, I-A^(b) (MHCII), CD40, CD80, CD86, CD11b, Gr1, CD49b, CD3,CD8, CD4, CD69, B220 or Foxp3 (BD Biosciences). Human monocytes werestained with fluorochrome-coupled antibodies to CD14 and CD3(eBioscience). Fluorescence data were collected on FACScalibur (BecktonDickinson) and analyzed using FlowJo software (Tree Star). For IL-12/p70measurement by ELISA (eBioscience), splenic DCs isolated as describedabove were cultured with or without CpG ODN for 18 h before collectingsupernatants.

ELISPOT Assay:

5×10⁵ cells isolated form tumor-draining lymph nodes of CpG- orODN-siRNAs-treated mice, were seeded into each well of 96-wellfiltration plate in the presence or absence of 10 μg/ml of p15E or TRP2peptide. After 24 h of incubation at 37° C., peptide-specificIFNγ-positive spots were detected according to manufacturer's procedure(Cell Sciences), scanned and quantified using Immunospot Analyzer fromCellular Technology Ltd.

Immunofluorescent and Intravital Two Photon Microscopy:

For immunofluorescent stanings, we fixed the flash-frozen tumorspecimens in formaldehyde, permeabilized with methanol and stained withantibodies to CD8, CD11b (BD Biosciences), TLR9 (eBiosciences), Dicer(Santa Cruz) and detected with Alexa488- or Alexa555-coupled secondaryantibodies from Invitrogen. After staining the nuclei with Hoechst 33342(Invitrogen), slides were mounted and analyzed by fluorescentmicroscopy. For intravital two-photon imaging, B16 tumor-bearing micereceived single intratumoral injection of 0.78 nmole FITC-labeledCpG-Stat3 siRNA, followed by retroorbital injection of dextran-rhodamine(Invitrogen) and Hoechst 33342 prior to imaging 2 h later. Mice wereanesthetized and intravital two-photon microscopy was carried out usingequipment and software from Ultima Multiphoton Microscopy Systems.

Statistical Analysis:

To compare tumor size or surface marker expression between multiple testgroups in animal experiments, we performed a one-way ANOVA followed byNewman-Keuls test. Unpaired t test was used to calculate two-tailed pvalue to estimate statistical significance of differences between twotreatment groups. Statistically significant p values were labeled asfollows: ***; p<0.001; **, p<0.01 and *, p<0.05. Data were analyzedusing Prism software (GraphPad).

Example 2 Stat3 Ablation in Hematopoietic Cells Drastically ImprovesTLR9 Triggering-Induced Antitumor Effects

To provide proof-of-principle evidence that targeting Stat3 can markedlyenhance CpG-ODN-based immunotherapy, we induced Stat3 allele truncationin the hematopoietic cells of adult mice using the Mx1-Cre-loxP systemas described previously (Kuhn et al., 1995). We employed PCR-basedgenotyping assay to confirm the truncation of loxP-flanked Stat3 allelesinduced by repeated injections of poly(I:C) in hematopoietic cells inMx1-Cre expressing mice. To avoid any interference from poly(I:C)treatment, subcutaneous B16(F10) tumor challenge was performed five daysafter last poly(I:C) administration. Established B16 tumors (day 10 post10⁶ tumor cell challenge, >10 mm diameter) were treated with a singleperitumoral injection of 5 μg CpG1668-oligonucleotide. Although CpG-ODNtreatment did not show significant antitumor activity in control mice(Stat3+/+) with heavy tumor load (FIG. 1 b—right panel), the sametreatment resulted in eradication of large B16 tumors (some of themreaching 1.5 cm in diameter) in mice lacking intact alleles within 3days after injection (FIGS. 1 a and 1 b). Similarly, whereas CpG-ODNinjection showed only weak inhibition of tumor growth in mice withsmaller initial B16 tumors (4-6 mm diameter) (FIG. 1 b—left panel),peritumoral treatment of with CpG-ODN in mice with truncated Stat3alleles in the hematopoietic cells resulted in regression of rapidlygrowing B16 tumors (FIG. 1 c) and prevented their reoccurrence over theperiod of at least 3 weeks (FIG. 1 d). In contrast, treatment withcontrol GpC oligonucleotide lacking CpG motif recognized by TLR9 did notsignificantly inhibit tumor progression.

To assess whether the dramatically increased antitumor effectscontributed by Stat3 inhibition in the hematopoietic cells was mediatedby T cells, we used CD8 and CD4 antibodies to deplete T cells. Theenhanced antitumor immunity due to Stat3 allele truncation inhematopoietic cells was abrogated in mice depleted of CD4 and CD8 Tcells (FIGS. 1 c and 1 d). However, lack of both lymphocyte populationsdid not prevent the initial robust tumor regression, strongly suggestingthe involvement of innate immunity in eliminating the establishedtumors. Indeed, NK cell depletion experiments indicated a partial roleof NK cells for the observed antitumor effect.

Example 3 Ablating Stat3 in Hematopoietic Cells Further Activates DCsPrimed by CpG

We next assessed if Stat3 inhibition affects CpG-induced DC activationin tumor-bearing mice. Flow cytometric analysis of CD11c+DCs isolatedfrom tumor-draining lymph nodes of mice with truncated Stat3 inhematopoietic cells showed enhanced DCs activation as measured byincreased expression of major histocompatibility complex (MHC) class II,CD86, CD80 and CD40 molecules two days after peritumoral injection ofCpG-ODN but not control GpC-ODN (FIG. 1 e—upper and lower panels). Wefurther assessed the expression of several immunostimulatory cytokineslike IL-12 (p35 and p40 subunits), RANTES and IL-6 in DCs freshlyisolated from tumor microenvironment following local treatment withCpG-ODN. As shown in FIG. 1 f, CpG-ODN induced high levels of all testedproinflammatory mediators in Stat3-deficient but not in wild-type DCswithin 18 hrs after treatment.

To evaluate the effect of Stat3 blocking on CpG-induced effectorlymphocyte activity, we analyzed CD8 T cells within tumor-draining lymphnodes of Stat3-positive and Stat3-negative mice after CpG-ODN treatment.CD8+ lymphocytes in tumor-draining lymph nodes of Stat3-ablated miceshowed very high levels of CD69 immediate early activation marker,shortly after peritumoral injection of CpG-ODN (FIG. 1 g, left panel).Moreover, 10 days after CpG-treatment Stat3-deficient mice had increasedability to mount tumor antigen-specific responses. The number ofIFN-γ-secreting T cells was strongly enhanced by CpG-treatment in thetumor-draining lymph nodes of Stat3−/− mice, as indicated by ELISPOTassay following ex vivo exposure to B16 tumor-specific p15E antigen(FIG. 1 g, right panel).

Example 4 Targeting Stat3 in Myeloid Cells (Dendritic Cells andMacrophages) by a Chimeric ssDNA-siRNA Construct

Results shown in FIG. 1 provide proof-of-principle evidence thatblocking Stat3 signaling in the hematopoietic cells removes a keynegative regulator of DC activation, thereby drastically improvingTLR-9-mediated DC activation and antitumor immunity. However, previousstudies indicated that prolonged, and effective blockade of Stat3signaling through gene ablation/truncation in the whole hematopoieticcompartment can lead to autoimmunity (Alonzi et al., 2004; Kobayashi etal., 2003; Welte et al., 2003). In order to minimize the side-effects ofStat3 blocking and yet achieve Stat3 inhibition-mediated enhancement ofantitumor immunity induced by TLR triggering, it would be ideal tospecifically and efficiently block Stat3 in antigen presenting cellswhile simultaneously activating TLR9 pathway. To achieve this goal, wetested the possibility to generate an ssDNA-dsRNA chimeric constructthat contains both CpG and Stat3 siRNA. The 20 bp long single-strandedCpG1668 ODN sequence was fused to a double-stranded 25/27-mer Stat3siRNA(FIG. 2 a). The selection of optimized 25/27 Stat3siRNA (both human andmouse) is based on the report that Dicer-processed siRNA has enhancedsilencing effects of target genes (Kim et al., 2005) (FIG. 7). In vitrocleavage assay confirmed that the chimeric CpG-Stat3 siRNA construct isprocessed by recombinant Dicer enzyme, just like the 25/27-mer Stat3siRNA without CpG (FIG. 2 a, lower panel).

To test cell specific uptake of chimeric ssDNA-dsRNA oligonucleotideconstructs, freshly isolated splenocytes from wild-type C57BL/6 micewere incubated overnight with the fluorescein-labeled CpG-Stat3-siRNAconstruct. Such incubation in the absence of any transfection agentsresulted in dose-dependent uptake of the DNA-RNA chimeric construct bysplenic DCs and macrophages but not granulocytes or T cells (FIG. 2 b).Under the same conditions the uptake of fluorescently-labeled nakedStat3-siRNA was insignificant. Further analysis of CpG-Stat3-siRNAuptake by confocal microscopy indicated rapid internalization of thechimeric construct with kinetics similar to the one previously reportedfor the CpG-ODN alone (Latz et al., 2004) (FIG. 2 c). In stable DC cellline (DC2.4), the CpG-Stat3-siRNA can be detectable as early as 15 min,with high uptake after 1 h of incubation. At this time pointCpG-Stat3-siRNA construct was found to colocalize with TLR9 withinperinuclear endocytic vesicles (FIG. 2 d—two top rows). Previous studiesindicated that binding of the Dicer nuclease to the siRNAoligonucleotide is required for its further processing into shorter21-mer fragments before interacting with RISC complex, which isresponsible for the final gene silencing effect (Chendrimada et al.,2005; Haase et al., 2005). We observed transient colocalization of theCpG-Stat3-siRNA with Dicer within 2 h after adding the oligonucleotidechimeric construct to cultured DCs. The association between theCpG-Stat3-siRNA and Dicer became weaker by 4 h and undetectable after 24h (FIG. 2 d—bottom two panels). These data suggest a sequential mode ofCpG-Stat3-siRNA construct action, which starts with the uptake intocytoplasmic endocytic vesicles, followed by binding to TLR9 andsubsequent interaction with Dicer. Quantitative real-time PCR analysisof the Stat3 mRNA expression in cultured primary DCs and in DC2.4 cellsindicated a dose-dependent downregulation of the Stat3 expression after24 h incubation with CpG-Stat3-siRNA, while the CpG-scrambled-RNAcontrol had negligible effect (FIG. 2 e). These results indicate thatchimeric CpG-siRNA molecules are efficiently internalized byTLR9-positive cells, undergo processing by Dicer and induce genesilencing. Of note, we observed that CpG treatment itself can upregulateStat3 activity and also gene expression (FIG. 2 f).

Example 5 In Vivo Characterization of the Chimeric CpG-siRNA

To evaluate the feasibility of using chimeric CpG-siRNA in vivo, weestimated first the uptake of the CpG-Stat3 siRNA in tumor-bearing mice.C57BL/6 mice with B16 tumors (6-10 mm in diameter) were injectedperitumorally with FITC-labeled CpG-Stat3 siRNA at 0.78 nmol (20μg)/injection. We detected large numbers of FITC-positive CD11b⁺ myeloidcells in tumor-draining nodes but not in collateral lymph nodes, 6 hafter injection (FIG. 3 a—top panel). More sensitive detection bytwo-photon microscopy confirmed the presence of FITC-positive cells intumor-draining lymph node as early as 1 h after injecting the labeledconstruct (FIG. 3 a—lower panel). Further studies have shown thatrepeated peritumoral and to lesser extent intravenous injections of 0.78nmole CpG-Stat3-siRNA, but not CpG-scrambled-RNA, silence Stat3expression in DCs and/or macrophages within tumor-draining lymph nodes(FIGS. 3 b and 3 d).

Example 6 Antitumor Effects of the CpG-Stat3 siRNA Chimeric Construct

Both macrophages and DCs in the tumor microenvironment are known topromote immune tolerance. We next assessed if the CpG-Stat3-siRNAchimeric constructs would negate immunosuppressive effects imposed bythe tumor-microenvironment and at the same time allow effectiveantitumor immunity induced by TLR9 triggering. Local treatment withCpG-Stat3-siRNA oligonucleotides inhibited growth of subcutaneouslygrowing B16 melanoma. In contrast, treatment with CpG-ODN alone or theCpG-scrambled-RNA construct had relatively weak antitumor effects (FIG.3 c). The ability of CpG-Stat3-siRNA to inhibit metastatic tumor growthwas further demonstrated in the model of established B16 lungmetastasis. We assessed the effect of 2-week systemic treatment withCpG-Stat3-siRNA, using relatively small amount of the oligonucleotide (1mg/kg). Systemic injection of 0.78 nmole CpG-Stat3-siRNA led tosignificant reduction in the number of lung metastasis with lessereffect of CpG-scrambled-RNA and CpG-ODN alone (FIG. 3 e), which isaccompanied by upregulation of MHC class II, CD80 and CD40 molecules ontumor infiltrating DCs (FIG. 3 f).

The ratio of effector to negative regulatory T cells within tumormicroenvironment is considered an important indicator of the effect ofadaptive immune responses against tumor. We investigated the numbers oftumor infiltrating T cell populations in subcutaneously growing B16tumors treated locally for 2 weeks with CpG-Stat3-siRNA,CpG-scrambled-RNA control or left untreated (FIG. 3 g). We observed anincrease in the infiltration of CD8+ T cells in the tumor stroma from 5to more than 20%, and an increase in tumor antigen, TRP2, positive CD8+T cells in the tumor (FIG. 3 h). In addition to CD8+ T cells, thenumbers of tumor-infiltrating NK cells and neutrophils are higher inmice treated with CpG-Stat3-siRNA (FIG. 3 g). Concomitant with anincrease in tumor infiltrating CD8+, NK and neutrophils that areimportant killing tumor cells, the percentage of CD4+/FoxP3+ T reg cellswithin all CD4+ T cells dropped from approximately 60 to 25% afterrepeated peritumoral injections of CpG-Stat3-siRNA (FIG. 3 g).

Example 7 Silencing STAT3 in Human Monocyte-Derived DCs to PreventImmunosuppression

The expression of TLR9 as well as the ability to take up CpG-basedoligonucleotides is reportedly restricted to relatively rare populationof plasmacytoid DCs in humans. However, moderate levels of TLR9expression have recently been found also in more common monocyte-derivedDCs (MoDCs) isolated or expanded from peripheral blood mononuclear cells(PBMCs). We created an analogue chimeric oligonucleotide by fusion ofCpG(D19) sequence optimized for activation of human TLR9-positive cellswith the STAT3-specific siRNA selected for the highest silencing effectin human cells. Next, we incubated fluorescein-labeledCpG(D19)-STAT3-siRNA with human PBMCs to determine the level andspecificity of oligonucleotide uptake (FIG. 4 a). Flow cytometricanalysis revealed the internalization of fusion oligonucleotide by CD14+monocytes but not by other PBMCs including CD3+ lymphocytes. Similarlyto the mouse DCs, CpG(D19)-STAT3-siRNA uptake is detectable after shortincubation time. Chimeric oligonucleotide internalization is dosedependent within the range of 20 to 500 nM, with maximal near 100%uptake at the highest concentration after 24 h (FIGS. 4 b and 4 c).Under these conditions, CpG(D19)-STAT3-siRNA reduced STAT3 expression byalmost 75% comparing to CpG-scrambled-RNA control as measured byreal-time PCR analysis (FIG. 4 d).

Example 8 Targeting Stat3 in Malignant B Cells by CpG-Stat3 siRNA

Not only Stat3 is activated in immune cells in the tumormicroenvironment, promoting tumor immunosuppression, Stat3 isconstitutively activated in tumor cells of diverse origin (Yu and Jove,2004; Yu et al., 2007). Stat3 activity intrinsic to the tumor cellsupregulate a large range of genes critical for tumor growth, survival,angiogenesis/metastasis and immunosuppression. It is therefore highlydesirable for any Stat3 inhibitor to be able to block Stat3 in the tumorcells. Because many malignant cells of B cell origin, including multiplemyeloma and B cell lymphoma express TLR9 (Bourke et al., 2003, Reid etal., 2005; Jahrsdorfer et al., 2005), we test the possibility thatCpG-Stat3siRNA can be internalized by these tumor cells, leading to genesilencing and tumor growth inhibition. To directly test thesepossibilities, we incubated several human B lymphoma cell lines withCpG-Stat3siRNA for uptake and internalization. The data shown in FIGS. 5a-5 e shows that CpG-STAT3 siRNA allows for siRNA delivery into varioustypes of human B lymphoma cells, in a dose-dependent manner. We thenassess the uptake and the effects of CpG-Stat3siRNA in a mouse myelomamodel. The results shown in FIGS. 6 a-6 c indicate that mouse myelomacells, MCP11, internalize FITC-labeled CpG-Stat3 siRNA in adose-dependent manner, as shown by flow cytometry after 24 h incubation.Furthermore, CpG-Stat3siRNA can lead to Stat3 silencing in MPC 11 cellstreated with 100 nM CpG-Stat3 siRNA for 24 h, as measured by real-timePCR. Importantly, MCP11 cells treated with CpG-Stat3siRNA leads toaccumulation in the G₂M phase of cell cycle as measured by flowcytometry after propidium iodide staining.

We next determined whether targeting Stat3 by CpG-Stat3 siRNA causesantitumor effects against MPC11 multiple myeloma. Mice bearing largeMCP11 tumors (10-13 mm in diameter) are injected intratumorally with0.78 nmole of CpG-siStat3 or CpG-scrRNA and two more times every secondday. MPC11 tumor is very aggressive, but in vivo treatment withCpG-Stat3siRNA results in significant tumor growth inhibition (FIG. 6a). Analysis of the tumor samples indicate that CpG-Stat3siRNA increasestumor cell apoptosis. An increased percentage of DCs in tumor-draininglymph-nodes after CpG-Stat3 siRNA treatment is also detected (FIG. 6 b).Moreover, there is an increase in CD40 and CD86 expression intumor-draining lymph node DCs (FIG. 6 c), suggesting that CpG-Stat3siRNAtreatment can lead to activation of DCs in the tumor milieu. Theseresults demonstrate that CpG-siRNA approach can target tumor cells of Bcell origin, leading to antitumor effects through direct effects on thetumor cells.

Example 8 Use of Activating RNAs to Promote Specific Gene Expression InVitro and In Vivo

Recently, it has been shown in several cases that double stranded smallRNA against targets at the promoter regions positively regulate genetranscription. For example, transcriptional activation of E-cadherin andVEGF is achieved by 21-nt double stranded RNAs targeting the promoterregion of these genes in human prostate cancer cells. We employed thesame method to activate mouse Edg1 gene, which is important forangiogenesis and immunosuppression. The small double strand RNA (21mer)sequences we identified for the mouse Edg1 gene are:

Sense:  (SEQ ID NO: 10) 5′ UGUCCUCUGUCCUCUAAGAUU-TT 3′ Antisense: (SEQ ID NO: 11) 5′ AAUCUUAGAGGACAGAGGACA-TT 3′

This Edg1 double stranded RNA when transfected into cells (both 3T3fibroblasts and B16 melanoma tumor cells) can induce strongtranscription of the Edg1 gene (FIG. 9).

Example 9 dsRNA Against Promoter Region of Edg1 Activates Edg1Transcription 3T3 Fibroblasts or B16 Melanoma Cells

Tumor cells transfected with the dsRNA against promoter region of Edg1,when implanted into mice, maintain high levels of Edg1 expression for atleast 3 weeks, as determined by analyzing tumors using real-time PCR(FIG. 10) at three weeks after tumor implantation.

The upregulation of Edg1 due to its own activating RNA leads toangiogenesis, immunosuppression and drastic tumor growth.

Examples 8 and 9 illustrate the use of short (21mer) double strandedRNAs to activate specific genes in vivo to modulate biologicalresponses, thereby leading to therapeutic outcomes. Linking CpG andother toll-like receptor ligand(s) with short siRNA illustrated in theprevious Examples is useful for targeted delivery of activating RNA,which like siRNA, is short double stranded RNA.

Example 10 Chemical Synthesis of Constructs Containing a TargetingMolecule and siRNA

The constructs that were synthesized consisted of the RNA sequence, CpGsequence, and of the linker connecting those two. Synthesis wasconducted on Perseptive Biosystems DNA Synthesizer Expedite 8909 in thetrityl-off mode.

Reagents: 5′-dimethoxytrityl-N-benzoyl-adenosine,2′-O-TBDMS-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;5′-dimethoxytrityl-N-acetyl-cytidine,2′-O-TBDMS-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;5′-dimethoxytrityl-N-isobutyryl-guanosine,2′-O-TBDMS-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;5′-dimethoxytrityl-uridine,2′-O-TBDMS-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;5′-dimethoxytrityl-N-benzoyl-deoxyadenosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;5′-dimethoxytrityl-N-acetyl-deoxycytidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;5′-dimethoxytrityl-N-p-tert-butylphenoxyacetyl-deoxyguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;and,5′-dimethoxytrityl-thymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramiditewere purchased from Azco Biotech, Inc. (San Diego, Calif., USA).

C3 spacer(3-(4,4′-dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)was synthesized in-house (Seela and Kaiser, 1987) or purchased from GlenResearch (Sterling, Va. USA). Ethylthiotetrazole (AIC) was used asactivator. Fluorescein phosphoramidite(1-dimethoxytrityloxy-2-(N-thiourea-(di-O-pivaloyl-fluorescein)-4-amino-butyl)-propyl-3-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite)(Glen Research, Sterling, Va., USA) was used for the introduction offluorescein into the oligomers.

Beaucage Reagent from American International Chemical, Inc. (AIC;Framingham, Mass., USA) was used for thioation of the phosphates of theCpG part of the construct.

The oligonucleotides were deprotected after synthesis with 10 Mmethylamine in ethanol-water 1:1, 55° C. for 30 min.

The deprotected constructs were purified by Polystyrene Reverse PhaseIon-Pair Chromatography (PRP-IPC). (Swiderski et al., 1994). Combinedfractions containing the pure product were concentrated under thereduced pressure to the volume of 1 ml. Ammonium acetate (100 mg) and2.5 ml of ethanol were added. Samples were kept at −20° C. for 4 hrs andthen centrifuged for 5 min. Supernatant did not have absorption at 260nm. Precipitate was resuspended in 1 ml of sterile water andre-precipitated as above. Preparative purification of oligonucleotideswas carried out on a Gilson Gradient HPLC System equipped in UniPointSystem Software. Purification was performed by Ion-Paired HPLC onpolystyrene resign PRP-1 (Hamilton) (4.6×250 mm); buffer A, 10 mMtetrabutylammonium acetate, water-acetonitrile 9:1 (pH 7.2); buffer B,10 mM tetrabutylammonium acetate, water-acetonitrile, 1:9, gradient0-65% of B in 30 min.

Analytical polyacrylamide gel electrophoresis (PAGE) was carried outusing 20% crosslinked gels (1 mm thick, 19:1 acrylamide:bis-acrylamide).Buffer: 100 mM Tris-borate, 1 mM EDTA, 7 M urea, pH 8.3 (25). Gels werevisualized by UV (254 nm) shadowing followed by methylene blue staining.Large scale synthesis of very pure, long (40-mers and longer) andcomplex oligonucleotides has its limitations. Due to the presence of RNAcomponent yields are lower and purification process more difficult.

TLR Agonist—Stat3 siRNA Conjugates Cell-Specific Gene Silencing andEnhanced Antitumor Immune Responses

Efficient delivery of siRNA to specific cell populations in vivo isimportant to its successful therapeutic application. Described inExamples 11-15 is a novel siRNA-based approach—synthetically linkingsiRNA to an oligonucleotide TLR9 agonist—that targets and silences genesin TLR9⁺ myeloid cells and B cells, both of which are key components ofthe tumor microenvironment is described. Because Stat3 intumor-associated immune cells suppresses antitumor immune responses andhinders TLR9 signaling, we tested CpG-Stat3siRNA conjugates foranti-tumor effects. When injected locally at the tumor site orsystemically through an intravenous route, the CpG-Stat3siRNA conjugatesaccess tumor-associated dendritic cells, macrophages and B cells,inhibit Stat3 expression, leading to activation of diversetumor-associated immune cells, and ultimately potent anti-tumor immuneresponses. The findings described herein demonstrate the potential ofTLR agonist-siRNA conjugates for targeted gene silencing coupled withTLR stimulation and immune activation in the tumor microenvironment.

Because Stat3 also restrains TLR-mediated Th1 immune responses(Kortylewski et al., 2005b; Kortylewski et al., 2009b; Yu et al., 2007),we reasoned that simultaneously silencing Stat3 by siRNA and triggeringTLRs by their agonists could effectively shift the tumormicroenvironment from pro-carcinogenic to anti-carcinogenic, potentiallyresulting in systemic tumor-specific immunity that could further inhibittumor metastases. A recent study using polymer-mediated in vivotransfection of 5′-triphosphate-Bcl2 siRNA has demonstrated the power ofsimultaneously inducing antitumor immunity and silencing an oncogenicgene (Poeck et al., 2008).

In this study, we explored a strategy of linking siRNAs to syntheticoligonucleotide agonists for endosomal TLRs, which include TLR3, TLR7,TLR8 and TLR9 (Iwasaki and Medzhitov, 2004; Kanzler et al., 2007;Barchet et al., 2008), for targeted delivery of siRNA into the endosomalcompartment of immune cells, such as myeloid cells and B cells, togetherwith TLR-dependent activation of antitumor immune responses. Theendosomal location of the oligonucleotide-binding TLRs, such as TLR9,might be advantageous in facilitating ultimate uptake of the siRNAcomponent to the cytosol of targeted cells for more efficient genesilencing in cells selectively expressing the cognate TLR. In order tomodel this concept, we chose TLR9-specific oligodeoxynucleotidescontaining an unmethylated CpG-motif (CpG ODN), because they are alreadyin clinical testing (Krieg, 2008). Additionally, CpG ODN are efficientlyinternalized by various antigen-presenting cells, such as dendriticcells, macrophages and B cells, and their binding to TLR9 can initiate acascade of innate and adaptive immune responses (Klimman et al., 2004;Barchet et al., 2008; Krieg, 2008). These immune cells are also criticalcomponents of the tumor microenvironment that actively promoteoncogenesis (Kujawski et al., 2008; Yu et al., 2007; Kortylewski et al.,2008; Bollrath et al., 2009; Grivennikov et al., 2009). By linking thesingle-stranded CpG ODN with double-stranded siRNA, we have created asingle synthetic molecule capable of delivering siRNA into myeloid and Bcells, silencing an immune checkpoint and/or oncogenic gene, andactivating TLR, leading to therapeutic antitumor immune responses.

Example 11 Materials and Methods for Examples 11-15

Cells:

Murine B16 melanoma, CT26 colon carcinoma and A20 B cell lymphoma lineswere purchased from American Type Culture Collection. Mouse dendriticDC2.4 cells were originally received from Dr. Kenneth Rock (Universityof Massachusetts Medical School, MA). Highly metastatic clone of K1735melanoma (C4) was obtained from Drs. S. Huang and J. Fidler of M. D.Anderson Cancer Center (Houston, Tex.). The stably transduced A20-Luccell line was kindly provided by Dr. Defu Zheng (City of Hope, Duarte,Calif.). The generation of transgenic C57BL/6.CEA mice and MC-38.CEAcell line was previously described (Tan and Coussens, 2007).

Oligonucleotide Design and Synthesis:

The phosphothioated oligodeoxyribonucleotide (ODN) and antisense strands(AS) of siRNAs were linked using 6 units of C3 carbon chain linker,(CH₂)₃ (Glen Research, Sterling, Va.). The resulting constructs werehybridized with complementary sense strands (SS) of siRNAs to createchimeric ODN-siRNA constructs used in the study (deoxynucleotides areshown underlined). Sequences of single stranded constructs are listedbelow.

Mouse Stat3 siRNA (SS) (SEQ ID NO: 3) 5′ CAGGGUGUCAGAUCACAUGGGCUAA 3′CpG1668-mouse Stat3 siRNA(AS) 5′ TCCATGACGTTCCTGATGCT-linker-UUAGCCCAUGUGAUCUGACACCCUGAA 3′ (SEQ ID NO: 1-linker-SEQ ID NO: 2)GpC-mouse Stat3 siRNA (AS) 5′ TCCATGAGCTTCCTGATGCT-linker-UUAGCCCAUGUGAUCUGACACCCUGAA 3′ (SEQ ID NO: 6-linker-SEQ ID NO: 2)Scrambled RNA (SS) (SEQ ID NO: 8) 5′ UCCAAGUAGAUUCGACGGCGAAGTG 3′CpG1668-scrambled RNA (AS) 5′ TCCATGACGTTCCTGATGCT-linker-CACUUCGCCGUCGAAUCUACUUGGAUU 3′ (SEQ ID NO: 1-linker-SEQ ID NO: 9)

The sequence of firefly luciferase-specific 25/27mer siRNA (Luc1 R25D/27), used for the CpG1668-Luc siRNA conjugate molecule, waspublished before (Rose et al., 2005). The correct formation of siRNAduplex was confirmed by in vitro Dicer cleavage assays. 0.5 μg of eachODN-siRNA construct was subjected to processing by 1 U of Dicer (Ambion)in 37° C. for 1.5 hr, resolved with 15% polyacrylamide/7.5M urea gel andresults of the dicing reaction were visualized with SYBR Gold staining(Invitrogen).

Quantitative Real-Time PCR:

Total RNA was extracted from cultured or primary cells using RNeasy kit(Qiagen). After cDNA synthesis using iScript cDNA Synthesis kit(Bio-Rad), samples were analyzed using pairs of primers specific forStat3, TNF, IL-6, IP-10, RANTES, p35/IL-12, p40/IL-12 and GAPDH mRNAs(SuperArray Bioscience Corporation). Sequence-specific amplification wasdetected by fluorescent signal of SYBR Green (Bio-Rad) by using Chromo4Real-time PCR Detector (Bio-Rad).

Transient Transfections:

B16 cells were transiently transfected with 15 nM CpG-linked oruncoupled Stat3 siRNA and scrambled RNA using Lipofectamine 2000 reagent(Invitrogen). 48 h later cells were lysed and used for western blot.

Electromobility Shift Assay (EMSA) and Western Blot:

EMSA and western blot analyses to detect Stat3 DNA-binding and proteinexpression were performed as described previously (Wang et al., 2004).The protein levels of Stat3 detected by western blotting were laterquantified by densitometry using AlphaEase FC software (Alpha Innotech).

Luciferase Reporter Gene Assay:

A20-Luc cells incubated with CpG-RNAs for 48 h or primary cells isolatedfrom tumor-draining lymph nodes of Luc⁺ mice treated with CpG-Luc siRNAwere lysed and luciferase activities were determined using theLuciferase Assay System (Promega) after normalization to the proteincontent of the sample.

In Vivo Experiments:

Mouse care and experimental procedures were performed underpathogen-free conditions in accordance with established institutionalguidance and approved protocols from Research Animal Care Committees ofthe City of Hope. For s.c. tumor challenge, we injected 1×10⁵ B16, C4 orCT26 tumor cells into 7-8 weeks C57BL/6, C57BL/6.CEA, C3H or Balb/Cmice, respectively. After tumors reached average size of ca. 5 mm, micewere injected peritumorally with 0.78 nmol of CpG1668 ODN alone, incombination with Stat3 siRNA or CpG/GpC ODN linked to various doublestranded RNA (dsRNA) sequences described above. Tumor growth wasmonitored every other day. For the analysis of cellular and molecularmechanisms of CpG/GpC-dsRNAs effects, mice were killed after 2-3 weeksof treatment. For experiments on silencing of luciferase activity invivo, Luc⁺ mice (originally kindly provided by Dr. Christopher H.Contag, Stanford University School of Medicine, CA) were challenged withtumor and treated with 3 daily injections of CpG-Luc siRNA orCpG-scrambled RNA. Lymph nodes as well as tumor specimens wereharvested. In experiments on B16 lung metastasis model, mice receivedintravenous injection of 0.5×10⁵ B16 tumor cells and two days later,after tumors were established, started to be treated systemically with0.78 nmol (ca. 1 mg/kg) of CpG1668 ODN alone or various CpG-dsRNAs.Intravenous injections were repeated every other day for two weeks.Lungs were harvested, fixed and the number of B16 colonies was manuallycounted. The level of Stat3 silencing was assessed by real-time PCR inDCs isolated from tumor-draining inguinal (for s.c. tumor model) orcervical (for metastasis model) lymph nodes. For immune cell depletion,mice were pretreated with anti-CD8 plus anti-CD4 antibodies (clone 2.43and GK1.5, respectively, depleting 99% and 98% of CD8 and CD4 cells,respectively) or anti-asialo-GM1 antibodies (Wako, depleting min. 79% ofNK cells) before tumor inoculation then injected weekly.

Flow Cytometry and ELISA:

We prepared single cell suspensions of spleen, lymph node or tumortissues by mechanic dispersion followed by collagenase D/DNase Itreatment as described before (Kortylewski et al. 2005b). Forextracellular staining of immune markers 1×10⁶ of freshly prepared cellssuspended in PBS/2% FCS/0.1% w/v sodium azide was preincubated withFcγIII/IIR-specific antibody to block non-specific binding and stainedwith different combinations of fluorochrome-coupled antibodies to CD11c,I-A^(b) (MHCII), CD40, CD80, B220, CD11b, Gr1, CD3, CD8 or CD4 (BDBiosciences). Prior to intracellular staining with antibodies to TLR9(eBioscience), Dicer (Santa Cruz) or FoxP3 (eBioscience), various immunecell subsets were fixed in paraformaldehyde and permeated in methanol.Fluorescence data were collected on FACScalibur (Beckton Dickinson) andanalyzed using FlowJo software (Tree Star).

ELISPOT Assay:

5×10⁵ cells isolated form tumor-draining lymph nodes of CpG- orCpG-siRNAs-treated mice, were seeded into each well of 96-wellfiltration plate in the presence or absence of 10 μg/ml of TRP2 peptide.After 24 h of incubation at 37° C., peptide-specific IFNγ-positive spotswere detected according to manufacturer's procedure (Cell Sciences),scanned and quantified using Immunospot Analyzer from CellularTechnology Ltd.

Fluorescent, Confocal and Intravital Two Photon Microscopy:

For immunofluorescent stanings, we fixed the flash-frozen tumorspecimens in formaldehyde and permeabilized with methanol beforeantibody staining. For confocal microscopy, cultured cells were fixedwith 2% paraformaldehyde for 20 min, permeabilized in PBS/0.1% TritonX-100/1 mM MgCl₂, and 0.1 mM CaCl₂ for 5 min and quenched in 50 mM NH₄Clfor 5 min before blocking in 1% BSA for 1 hr. Samples were stained withantibodies to CD11b (BD Biosciences), neutrophils (7/4, Cedarlane),active caspase-3 (Cell Signaling), TLR9 (eBiosciences), Dicer (SantaCruz) and detected with Alexa488- or Alexa555-coupled secondaryantibodies (Invitrogen). After staining the nuclei with DAPI (Vector) orHoechst 33342, slides were mounted and analyzed by fluorescent orconfocal microscopy. The confocal imaging was carried out using a 63×water immersion objective on cLSM510Meta confocal microscope (Zeiss).For intravital two-photon imaging, B16 tumor-bearing mice receivedsingle intratumoral injection of 0.78 nmol FITC-labeled CpG-Stat3 siRNA,followed by retroorbital injection of dextran-rhodamine (Invitrogen) andHoechst 33342 prior to imaging 1 h later. Mice were anesthetized andintravital two-photon microscopy was carried out using equipment andsoftware from Ultima Multiphoton Microscopy Systems.

Statistical Analysis:

Unpaired t-test with equal or unequal variance (specifically for theanalysis of cytokine expression in FIG. 26 a) was used to calculatetwo-tailed P value to estimate statistical significance of differencesbetween two treatment groups in the whole study. One-way ANOVA followedby Newman-Keuls test was applied for comparison of multiple treatmentgroups. Two-way ANOVA plus Bonferroni posttest were applied to assessstatistical significance of differences in tumor growth kinetics betweenmultiple treatment groups. Statistically significant P values wereindicated in figures and/or legends and labeled as follows: ***;P<0.001; **, P<0.01 and *, P<0.05. Data were analyzed using GraphPadPrism vs. 4.0 software (GraphPad).

Example 12 Construction and In Vitro Characterization of the Cpg-Stat3siRNA Conjugate Molecule

Synthesis of the antisense strand of the siRNA (27mer) was followed byCpG1668 ODN synthesis (Klinman et al., 19996; Krieg et al., 1995),producing a single stranded oligonucleotide connected through a carbonchain linker. The sense strand sequence of the Stat3 siRNA (25mer) isalso shown (FIG. 11 a). A 25/27mer form of siRNA was chosen over theconventional 21mer duplex to allow uncoupling of the siRNA from the CpGsequence by the Dicer enzyme once inside the cell. The asymmetric25/27mer siRNA was optimized for specific processing by Dicer and wasmore potent in silencing of target genes (Kim et al., 2005; Rose et al.,2005). We first evaluated whether attaching siRNA to CpG ODN through thelinker would interfere with TLR9 activation. Adding either CpG1688 aloneor CpG-Stat3 siRNA conjugate to cultured DC2.4 dendritic cells resultedin a similar increase in expression of co-stimulatory CD40 and CD80molecules, suggesting that CpG-Stat3 siRNA retains its capacity toactivate TLR9 (FIG. 11 b). In addition, we verified that theimmunostimulatory properties of CpG-siRNA conjugates do not differ fromthe effect of CpG alone as measured by production of inflammatorycytokines in primary cells and NF-κB/AP1 activation in a stablemacrophage cell line designed for such test (FIGS. 12 a-12 d). To assesswhether the conjugation of siRNA with CpG moiety would still allow Dicerprocessing, we compared in vitro Dicer activity on CpG-Stat3 siRNAsubstrate versus 25/27mer Stat3 siRNA alone. The CpG-Stat3 siRNA andStat3 siRNA were incubated with 1 U of recombinant Dicer for 1 h at 37°C. and then visualized on polyacrylamide gel through SYBRGold staining.Both the CpG-Stat3 siRNA and Stat3 siRNA were processed to 21mer siRNAby recombinant Dicer (FIG. 11 c). Finally, to determine whether theCpG-Stat3 siRNA retains gene silencing function, the chimeric moleculewas transfected into cells using Lipofectamine transfection reagent.Results from this experiment indicated that linking CpG ODN to siRNA didnot interfere with Stat3 gene silencing (FIG. 11 d). Both CpG-Stat3siRNA and Stat3 siRNA alone reduced the total protein levels of Stat3 by55% and 49%, respectively, as measured by densitometry.

Example 13 Cell Specific Uptake and Gene Silencing Effects by theCpG-siRNA Conjugate Molecules

To determine the specificity and efficiency of CpG-siRNA uptake, freshlyprepared mouse splenocytes were incubated for 3 h with twoconcentrations of CpG-linked Stat3 siRNA or an unconjugated Stat3 siRNA,in the absence of any transfection reagent(s). Both the CpG-Stat3 siRNAand unconjugated Stat3 siRNA were labeled with fluorescein.Fluorescein-positive DCs, macrophages, B cells, granulocytes and T cellswere assessed by FACS analysis. Results from the flow cytometricanalyses indicated that the chimeric CpG-Stat3 siRNA was efficientlytaken up by both plasmacytoid (CD11c⁺B220⁺) and conventional(CD11c⁺B220⁻) splenic DCs, macrophages (F4/80⁺Gr1⁻) and B cells(B220⁺CD11c⁻), whereas uptake by splenic granulocytes (Gr1⁺F4/80⁻) or Tcells (CD3⁺) was minimal (FIG. 13 a, FIG. 14 and Table 1). This uptakepattern reflects the known distribution of TLR9 expression in murineleukocyte subsets (Hemmi et al., 2000; Iwasaki and Medzhitov, 2004).CD11c⁺ DCs were confirmed to express TLR9, as shown by intracellularstaining of TLR9 in fixed splenocytes by flow cytometry (FIG. 13 a,bottom right). Unconjugated Stat3 siRNA was not efficiently incorporatedinto DCs even after 24 h incubation, demonstrating that linkage to theTLR9 ligand facilitates siRNA uptake (FIG. 13 a, bottom left).

TABLE 1 Untreated 100 nM 250 nM 500 nM mDCs 0% 75% 94% 97% pDCs 0% 83%96% 98% MCs 0% 75% 91% 96% B cells 0% 22% 64% 94% Granulocytes 0% 16%28% 29% T cells 0%  9% 16% 16%

We further evaluated CpG-Stat3 siRNA-FITC uptake by DC 2.4 mousedendritic cells. FACS analyses and fluorescent microscopy indicated thatwithout transfection reagents, the CpG-Stat3 siRNA-FITC was internalizedby DC 2.4 cells, with kinetics similar to that CpG-ODN alone andreported previously (Latz et al., 2004) (FIG. 13 b—two top rows and FIG.15). By 60 min, greater than 80% DC 2.4 cells were positive for uptakeof the conjugate, which was confirmed by confocal microscopic analysis.The uptake of the CpG-Stat3 siRNA-FITC was dose dependent, observable atthe concentration of 100 nM and reaching maximum at 500 nM (FIG. 13 b,bottom row).

Confocal microscopy analyses further showed that at one hour afterincubation, the CpG-Stat3 siRNA colocalized with TLR9 within perinuclearendocytic vesicles (FIG. 13 c, two top rows; and FIG. 16). Thiscolocalization diminished at 2 and 4 h after CpG-siRNA treatment (FIG.13 c, two top rows. Previous studies have demonstrated that binding ofthe Dicer nuclease to the siRNA oligonucleotide is required for furthersiRNA processing to shorter 21mer fragments that can mediate RISCcomplex-dependent mRNA degradation (Chendrimada et al., 2005). Weobserved transient colocalization of the CpG-Stat3 siRNA with Dicerwithin 2 h after adding the oligonucleotide to cultured dendritic cells(FIG. 13 c, two bottom rows and FIG. 16). The colocalization ofCpG-Stat3-siRNA and Dicer became weaker by 4 h (FIG. 13 c) andundetectable after 24 h (data not shown).

To determine gene silencing effects of the CpG-Stat3 siRNA, DC2.4 cellswere incubated with CpG-Stat3 siRNA, CpG-scrambled RNA or GpC-conjugatedStat3 siRNA. Quantitative real-time PCR analysis of the Stat3 mRNAexpression in DC2.4 cells indicated a dose-dependent downregulation ofStat3 expression by CpG-Stat3 siRNA, compared to CpG-scrambled RNA (FIG.13 d). Maximum effect on Stat3 silencing (ca. 80% reduction) wasobserved at relatively high 1 μM concentration of CpG-Stat3 siRNA inserum-containing cell culture medium. GpC-conjugated Stat3 siRNA, whichbinds but does not activate TLR9 (Latz et al. 2004), failed to silenceStat3, suggesting a possible requirement of TLR9 activation for theCpG-siRNA to be further processed. Further experiments demonstrated thatin TLR9^(−/−) myeloid cells and DCs, while cellular uptake of CpG-Stat3siRNA was normal (FIG. 17), the gene silencing effect of CpG-siRNA wascompletely impaired (FIG. 13 e). We further confirmed the gene silencingeffects using electrophoretic mobility shift assays (EMSA) to detectStat3 DNA-binding activity in DC2.4 cells, which was induced by IL-10stimulation (FIG. 13 f). Note that, as indicated above, stimulationusing CpG itself also resulted in Stat3 activation, which serves as anegative feedback mechanism (Kortylewski et al., 2009b; Samarasinghe etal., 2006) (FIG. 13 f), thereby complicating the EMSA analysis fordetection of Stat3 silencing. None-the-less, the higher concentrationsof CpG-Stat3 siRNA diminished Stat3 DNA binding activity relative to theconjugate scrambled RNA controls.

To demonstrate the generality of this approach to gene silencing, weused another system to verify the gene silencing effects of theCpG-siRNA constructs. Mouse A20 B cell lymphoma cells, which areTLR9-positive, could internalize CpG-siRNA (FIG. 18 a). A chimericconjugate linking a Dicer substrate siRNA specific for fireflyluciferase (Luc) conjugated to the deoxyribonucleotides CpG1668 ODN(CpG-Luc siRNA) inhibited luciferase overexpression in A20-Luc cells,which was determined by measuring luciferase activity (FIG. 18 b).

Example 14 Antitumor Effects of the CpG-Stat3siRNA Conjugate Molecule

To evaluate the feasibility of using the CpG-siRNA conjugates in vivofor therapeutic purposes, we focused on potential immunologicallymediated anti-tumor effects of the CpG-Stat3 siRNA conjugates. Theinitial biodistribution experiments in naive tumor-free mice confirmedthat CpG-Stat3 siRNA is specifically internalized by residentmacrophages in different tissues as well as DCs and B cells in lymphnodes (FIG. 19). Next, we estimated the uptake of the CpG-Stat3 siRNA bymacrophages and dendritic cells in tumor-bearing mice. C57BL/6 mice withaggressive poorly immunogenic B16 tumors (6-10 mm in diameter) wereinjected peritumorally with FITC-labeled CpG-Stat3 siRNA at 0.78 nmol(20 μg)/injection. As shown by immunofluorescent staining and FACSanalysis, numerous myeloid cells accumulated at the site of CpG-Stat3siRNA injection already 1 h later (FIG. 20 a and FIG. 21 a).Furthermore, in vivo intravital two-photon microscopy indicated thepresence of FITC-positive cells in tumor-draining lymph node, as earlyas 1 h after injection of the labeled construct (FIG. 20 b, FIG. 21 band FIG. 22), but not in the contralateral lymph nodes (FIG. 22).Additionally, high resolution imaging by intravital two-photonmicroscopy revealed not only an increased number of FITC-positive cellsin tumor draining lymph nodes, but also an accumulation of FITC-labeledCpG-Stat3 siRNAs in perinuclear endocytic vesicles (FIG. 20 b, bottomright), which was also observed in cultured dendritic cells (FIG. 13 c).

We next evaluated the gene silencing and antitumor effects of CpG-Stat3siRNAs in vivo. Peritumoral injections of the CpG-Stat3 siRNA resultedin relatively effective gene silencing in dendritic cells, macrophagesand B cells accumulated in tumor draining lymph nodes, compared tocontrol CpG-Luc siRNA, as measured by quantitative real-time PCR (FIG.20 c). Stat3 inactivation in CD11c⁺ dendritic cells isolated from tumordraining lymph nodes was confirmed at protein level (FIG. 20 d).Furthermore, quantitative real-time PCR and Western blotting indicateStat3 silencing in the total tumor draining lymph nodes as well (FIG.23). We also used CpG-Luc siRNA conjugate to confirm that CpG-siRNAconjugates are able to reduce protein expression specifically withinmyeloid cells in vivo. Mice over-expressing firefly luciferase undercontrol of the β-actin promoter (Cao et al., 2004) were challenged withtumor cells, followed by repeated peritumoral injections of CpG-LucsiRNA. Results from these experiments indicated effective inhibition ofluciferase activity in CD11b⁺ myeloid cells but not in CD4⁺ lymphocyteswithin tumor-draining lymph nodes (FIG. 20 e).

Both dendritic cells and macrophages in the tumor microenvironment areknown to promote immune tolerance (Dhodapkar et al., 2008; Vicari etal., 2002; Zou et al. 2005). Our previous work demonstrated that Stat3is constitutively-activated in myeloid cells in the tumor milieu andthat genetic ablation of Stat3 in the myeloid compartment elicits potentantitumor immunity (Kortylewski et al., 2005b). Furthermore, both CpGand LPS treatment activates Stat3 (Samarasinghe et al., 2006;Kortylewski et al., 2009b; Benkhart et al., 2000), which acts as anegative feedback mechanism to constrain Th1 immune responses.Therefore, we assessed whether the CpG-Stat3-siRNA conjugates couldreverse the immunosuppressive effects imposed by thetumor-microenvironment and at the same time allow effective antitumorimmunity induced by TLR9 triggering. Local treatment with CpG-Stat3siRNA oligonucleotides inhibited growth of subcutaneously growing B16melanoma (3-5 mm in diameter at the initial treatment). In contrast,treatment with unconjugated CpG-ODN plus Stat3 siRNA, or CpG-scrambledRNA construct, or GpC-Stat3 siRNA had significantly less antitumoreffects (FIG. 24 a). This finding was confirmed by using two additionalCpG-Stat3 siRNA conjugates containing alternative Stat3 siRNA sequences(data not shown). To confirm that the antitumor effects induced byCpG-Stat3 siRNA were mainly mediated by immune cells, we performed invivo experiments with antibody-mediated depletion of CD8/CD4 T cells andNK cells. As shown in FIG. 24 b, in the absence of CD8⁺ and CD4⁺ immunecell populations (including possibly also the cross priming CD11c⁺CD8⁺DCs), the effects of CpG-Stat3 siRNA treatment were partially reducedand comparable to the moderate antitumor effect of the control CpG-LucsiRNA, and lack of NK cells abrogated therapeutic effect of CpG-Stat3siRNA.

We confirmed that local treatment with CpG-Stat3 siRNA can reduce growthof other tumors independently of their genetic background. CpG-Stat3siRNA oligonucleotides inhibited growth of both a poorly immunogenicvariant of K1735 melanoma, C4 (Xie et al., 2004), and CT26 coloncarcinomas in C3H and BALB/c mice, respectively (FIGS. 24 c, 24 d).Furthermore, CpG-Stat3 siRNA treatment of the carcinoembryonic antigen(CEA) transgenic C57BL/6 mice bearing MC38 colon carcinomas expressingCEA led to tumor regression (FIG. 24 e). To assess in vivo effects ofthe CpG-Stat3 siRNA on tumor cells, we stained B16 tumor tissues withfluorescent antibody specific to activated caspase-3. Data from theseanalysis showed that B16 tumors received CpG-Stat3 siRNA had undergonemore extensive apoptosis relative to the other three treatment groups(FIG. 25).

We further investigated the possibility that intravenous injections ofCpG-Stat3 siRNA can lead to gene silencing and antitumor effects.Intravenous injection of CpG-Stat3 siRNA (0.78 nmol) reduced Stat3expression in dendritic cells within tumor-draining lymph nodes relativeto CpG-scrambled RNA (FIG. 24 f). We also tested the ability of systemicdelivery of CpG-Stat3 siRNA to inhibit metastatic tumor growth in anestablished B16 lung metastasis model. Mice with disseminated B16 tumorcells were treated systemically with CpG-Stat3 siRNA thrice weekly fortwo weeks. Relatively small amounts of the oligonucleotide (<1 mg/kg)were used for the systemic injection, which led to significant reductionin the number of lung metastasis (FIG. 24 g). A significantly lowerantitumor effect due to CpG-scrambled RNA and CpG ODN alone was alsoobserved. Thus, maximal antitumor effects required conjugation of theCpG TLR9 ligand with a functional Stat3 siRNA.

Example 15 Modulation of the Tumor Immunologic Milieu by the CpG-Stat3siRNA Conjugate Molecule

To further assess the role of immune modulation in the observedantitumor effects mediated by CpG-Stat3 siRNA conjugate treatment, weanalyzed changes in Th1 cytokine/chemokines and co-stimulatory moleculeexpression by dendritic cells in the tumor draining lymph nodes. Lack ofStat3 in DCs has been shown to upregulate expression of Th1cytokines/chemokines (Kortylewski et al., 2005b; Kortylewski et al.,2009a; Takeda et al., 1999; Welte et al., 2003), which can be greatlyamplified by CpG treatment (Kortylewski et al., 2009b;). As shown inFIG. 26 a, local tumor site injection of the CpG-Stat3 siRNA resulted inupregulation of several Th1 cytokines and chemokines, which were shownto be upregulated by Stat3 ablation (Kortylewski et al., 2005b;Kortylewski et al., 2009a; Takeda et al., 1999; Welte et al., 2003). Ithas also been documented that dendritic cells with low expression levelsof co-stimulatory molecules mediate immune tolerance (Dhodapkar et al.,2001), which is one of the proposed mechanisms for tumor immune evasioninduced by Stat3 activation in tumor-associated dendritic cells(Kortylewski et al., 2005b). We therefore analyzed expression ofco-stimulatory molecules by dendritic cells enriched from tumor draininglymph nodes. Results from these analyses indicated that CpG-Stat3 siRNAreduced the number of the dendritic cells with low expression ofco-stimulatory molecules, including MHC class II, CD80 and CD40, whichwas accompanied by a modest increase in expression of theseco-stimulatory molecules (FIG. 27). Stat3 ablation in myeloid cellsfollowed by local treatment has been shown to induce potent antitumorinnate immune responses that involve neutrophils (Kortylewski et al.,2005b). We therefore assessed whether CpG-Stat3 siRNA conjugatetreatment could lead to neutrophil-associated tumor cell apoptosis.Co-staining B16 tumor tissue sections with antibodies specific toactivated caspase-3 and neutrophils revealed that CpG-Stat3 siRNAtreatment-induced massive tumor cell apoptosis (activated caspase3-positive) was associated with an increase in tumor-infiltratingneutrophils (FIGS. 28 a, 28 b).

The ratio of effector to regulatory T cells within the tumormicroenvironment is considered to correlate well with the effect ofadaptive immune responses on tumor progression and metastasis (Bui etal., 2006). We investigated the numbers of tumor infiltrating T cellpopulations in subcutaneously growing B16 tumors treated locally for 2weeks with CpG-Stat3 siRNA, CpG-scrambled RNA control or treated withPBS only (FIG. 24 a). We found that although CpG-Stat3 siRNA treatmentdid not induce significant changes in overall CD4⁺ T cell numbers withinthe tumors, as shown by flow cytometric analysis (FIG. 28 a), thepercentage of CD4⁺/FoxP3⁺ Treg cells within all CD4⁺ T cells droppedfrom approximately 60% to 25% after peritumoral injections of CpG-Stat3siRNA (FIG. 28 b). We observed an increase in the infiltration of totalCD8⁺ T cells in the tumor stroma from 5% to almost 20%, althoughCpG-scrambled RNA control treatment also led to some recruitment of CD8⁺T cells, as shown by flow cytometric analysis (FIG. 28 c). These effectsprobably result from TLR9-mediated immunostimulation oftumor-infiltrating APCs. At the same time, we cannot rule out antitumoreffects contributed by non-specific immunostimulation by double-strandedRNA. Fluorescent immunostaining of frozen tumor tissues with anti-CD8antibody confirmed data generated by flow cytometric analysis (FIG. 28c) that CpG-Stat3 siRNA treatment caused increased CD8⁺ T cellinfiltration in tumors (data not shown). Activation of tumorantigen-specific CD8⁺ T cells is believed to be critical forimmune-mediated antitumor effects. We therefore examined the ability ofCpG-Stat3 siRNA treatment to generate CD8⁺ T cells specific for the B16tumor antigen, TRP2. ELISPOT assays to determine IFNγ production by Tcells isolated from tumor draining lymph nodes in response to recallstimulation with TRP2 peptide indicated that in vivo CpG-Stat3 siRNAadministration indeed induced antigen-specific CD8⁺ T cells (FIG. 28 d).

Additional results are shown in FIGS. 29 a-29 c for different siRNAStat3 sequences. FIGS. 30 a-30 b also show that TLR9 is not necessaryfor uptake but is required for silencing effect of CpG-Stat3 siRNA bymyeloid cells. FIGS. 31 a-31 e show that the CpG-siRNA approacheffectively silences genes in TLR9⁺ human tumor cells leading totherapeutic antitumor effects in animals. These results (i) show thevalidation of CpG-siRNA approach for cancer immunotherapy by usingadditional siRNAs with different sequences; (ii) show that TLR9 is notnecessary for uptake but is required for silencing effect of CpG-Stat3siRNA by myeloid cells and (iii) show that the CpG-siRNA approacheffectively silences genes in TLR9⁺ human tumor cells leading totherapeutic antitumor effects in animals.

We have developed a new strategy for targeted siRNA delivery togetherwith immune activation by covalently linking TLR oligonucleotideagonists to siRNAs. These conjugates encompass three activities in asingle molecule: targeting to immune cells, which include DCs,macrophages, and B cells, TLR activation and immune checkpointsilencing. In addition to TLR9, several other intracellular TLRs, suchas TLR3, TLR7 and TLR8 also recognize nucleic acids, suggesting a broadapplication of this approach using various ligands for these TLRs todeliver various siRNAs into different immune cells. TLRs are importantfor stimulating dendritic cell maturation, antigen uptake andpresentation, leading to CTL activation and CD4⁺ T helper celldifferentiation. Therefore, TLR agonist-siRNA approaches can furtherstimulate desired immune responses for treating diseases such as cancerand infections. Although it has been established that binding to TLR9 isnecessary for CpG-mediated immune activation, it remains to be fullyexplored how CpG ODN enter cells (Latz et al., 2004). Our resultsindicated that cellular uptake of both CpG ODN and the CpG-siRNAconstructs can occur in the absence of TLR9. However, TLR9 is requiredfor CpG-siRNA mediated gene silencing. While the exact underlyingmechanism(s) remains to be determined, it is possible that triggeringTLR9 could effect either endosomal release of CpG-siRNA into thecytoplasm, or/and its intracellular processing.

Although TLR9 is expressed in different types of mouse dendritic cells,its expression is more selective in humans. While the highest levels ofconstitutive TLR9 expression is observed on human plasmacytoid DCs and Bcells, it is also expressed at lower levels on human monocytes andmacrophages (Iwasaki and Medzihitov, 2004). These immune cells can serveas antigen-presenting cells and induce innate, adaptive or humoralimmunity (Kanzler et al., 2007; Krieg, 2008; Marshner et al., 2005;Klinman et al., 2008). Furthermore, it has been demonstrated recentlythat adding triphosphate to the 5′ of siRNA can greatly potentiate theantitumor effects of siRNA by stimulating antitumor immune responses,likely through intracellular RNA sensors such as RIG-I or MDA-5 (Poecket al., 2008). It is therefore conceivable to incorporate triphosphateto the CpG-siRNA to further amplify antitumor immunity. In addition, acritical role of tumor stromal macrophages and B cells in promotingtumor development has been well documented (Pollard, 2004; Sica andBronte, 2007; Tan and Coussens, 2007). Importantly, Stat3 and severalother molecules produced by the tumor myeloid population, and possiblytumor-associated B cells, are critical for tumor immunosuppression (Yuet al., 2007), and Stat3 activity in the myeloid compartment (possibly Bcells as well) promotes Stat3 activity in tumor cells and endothelialcells in the tumor, enhancing tumor cell growth/survival (Kujawski etal., 2008; Bollrath et al., 2009; Grivennikov et al., 2009; Lee et al.,2009). In addition to Stat3, other oncogenic molecules produced by thetumor myeloid/B cell compartment are also critical in promoting cancergrowth and resistance to various therapies. Therefore, being able totarget the tumor stromal myeloid cells/B cells through CpG-siRNAconjugate molecules is highly desirable for cancer therapies. Inaddition to normal immune cells, several types of tumor cells, includingthose of B cell origin, and some solid tumor cells, are also positivefor TLR9 (Jahrsdorfer et al., 2005; Spaner et al., 2008). Ourpreliminary results suggested the feasibility of CpG-siRNA approach tosilence genes in TLR9⁺ tumor cells. For example, treating human TLR9⁺tumors in NOD/SCID/IL-2RγKO mice with CpG-Stat3 siRNA resulted in tumorcell apoptosis and tumor growth inhibition (Kortylewski and Yu,unpublished data).

Our results indicated that the gene silencing effects by CpG-siRNA incultured cells requires high concentrations of the conjugates and aresuboptimal relative to in vivo treatment. Work is underway to determinethe possible cause(s) of this difference, which might includeserum-dependent degradation of CpG-siRNAs or reduction of the overallsilencing effect in rapidly dividing cell populations. It is possiblethat in vivo repeated treatments allow accumulative gene silencingeffects, and the crosstalk between various cells in the tumormicroenvironment could lead to secondary effects on Stat3 activity (Leeet al., 2009). The half-life of the constructs at present is limited.Although the CpG ODN in the construct is phosphorothioated, which shouldresist serum degradation, the siRNA in the chimeric construct isunmodified and negatively charged. Chemically modifying the siRNA toprolong serum stability and to neutralize the negative charge of thesiRNA to facilitate endosomal release may improve the efficacy of TLRagonist-siRNA approach. Our results show the use of oligonucleotide TLRagonists for siRNA delivery into tumor-associated myeloid cells and Bcells to inhibit expression of tumor-promoting/immunosuppressivemolecules while activating TLR(s) for immune activation.

The proof-of-principle experiments provided evidence that systemicallydelivered CpG(A)-STAT3 siRNA (i.e., CpG(D19)-STAT3 siRNA) can targethuman TLR9⁺ cells in vivo. The intravenously injected CpG(A)-STAT3 siRNAled to STAT3 gene silencing in human MV4-11 acute myeloid leukemia (AML)cells residing in the bone-marrow of immunodeficientNOD/SCID/IL-2Rγ^(null) (NSG) mice (FIG. 32 a). In other preliminarystudies, effects of CpG(A)-siRNAs targeting oncogenic and/or prosurvivalgenes injected intratumorally into KMS-11 multiple myeloma (MM) (FIG. 32b) or MonoMac6 and MV4-11 AML (FIG. 32 c and FIG. 32 d) tumors growings.c. in NSG mice were compared. The repeated local administration ofCpG(A)-siRNAs specific for either STAT3 or BCL-X_(L) genes resulted ingene-specific silencing, induced tumor cell death and reduced growth ofxenotransplanted tumors (FIGS. 32 b-32 d).

In preliminary studies, the silencing efficacy of CpG-siRNA conjugatesbased on CpG oligodeoxynucleotides from either class A or class B werealso compared. As shown in FIGS. 33 (top) and 33 (bottom), class A-basedCpG(D19)-STAT3 siRNA induced more pronounced target gene silencingeffect (FIG. 33 (top)) and higher degree of tumor cell death (FIG. 33(bottom)) that than the class B-based CpG(7909)-STAT3 siRNA incomparison to matching control class A and class B CpG-LuciferasesiRNAs, respectively. Sequences of single stranded constructs notdisclosed above are listed below.

CpG(7909)-human STAT3 siRNA (antisense strand; underlined aredeoxyribonucleotides, asterisks indicate phosphothioated sites, Xindicates single C3 carbon chain linker)

5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T-XXXXX-UCAGUCGUAUCUUUCUGCAGCUUCCGU 3′ (SEQ ID NO: 21-XXXXX-SEQ ID NO: 5)

CpG(7909)-Luciferase siRNA (antisense strand; underlined aredeoxyribonucleotides, asterisks indicate phosphothioated sites, Xindicates single C3 carbon chain linker)

5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T-XXXXX-UGUAAAAGCAAUUGUUCCAGGAACCAG 3′ (SEQ ID NO: 21-XXXXX-SEQ ID NO: 22)

Luciferase siRNA (sense strand; underlined are deoxyribonucleotides)

(SEQ ID NO: 23) 5′ GGUUCCUGGAACAAUUGCUUUUACA 3′

To assess the potential safety of CpG-siRNAs for normal human immunecells, cultured human peripheral blood mononuclear cells (PBMCs) wereused. Multiplex assays indicated that CpG(D19)-STAT3 siRNA induced moredesirable cytokine expression profile comparing to the class B-basedCpG(7909)-STAT3 siRNA and class C-based CpG(2429)-STAT3 siRNA (FIG. 34).Although inflammatory cytokines/chemokines, such as IFNα, IP-10, MCP-1,were induced at similar levels by all three conjugates, both CpG(7909)-and CpG(2429)-STAT3 siRNAs also led to production of potentiallytumorigenic/tolerogenic IL-6 and IL-10 that could dampen the overallimmunostimulatory effect. Importantly, the in vitro studies on humanPBMCs demonstrated that CpG(A)-STAT3 siRNA was immunostimulatory but notimmunotoxic for normal immune cells. The siRNA conjugation to CpG(D19)eliminated the exacerbated interferon type I responses typical for classA of CpG ODNs (FIG. 35), which hindered their clinical application.Sequences of single stranded constructs not disclosed above are listedbelow.

CpG(2429)-human STAT3 siRNA (antisense strand; underlined aredeoxyribonucleotides, asterisks indicate phosphothioated sites, Xindicates single C3 carbon chain linker)

5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G-XXXXX-UCAGUCGUAUCUUUCUGCAGCUUCCGU 3′ (SEQ ID NO: 24-XXXXX-SEQ ID NO: 5)

CpG(2429)-Luciferase siRNA (antisense strand; underlined aredeoxyribonucleotides, asterisks indicate phosphothioated sites, Xindicates single C3 carbon chain linker)

5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G-XXXXX-GUAAAAGCAAUUGUUCCAGGAACCAG 3′ (SEQ ID NO: 24-XXXXX-SEQ ID NO: 22)

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

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What is claimed is:
 1. An oligonucleotide comprising a modified humanSTAT3 sense strand comprising an oligonucleotide having the nucleotidesset forth in SEQ ID NO:17, wherein nucleotides at positions 3, 4, 7, 9,10, 12-4, 16-19 and 23 are modified and wherein the modifications areindependently selected from the group consisting of a 2′-alkylpyrimidine, 2′F, 2′OMe, amino, a deoxynucleotide, an abasic sugar, a2-O-alkyl modified pyrimidine, 4-thiouracil, 5-bromouracil,5-iodouracil, 5-(3-aminoallyl)-uracil and LNA.
 2. A chimeric moleculecomprising a modified human STAT3 sense strand and a humanCpG(D19)-STAT3 antisense strand, wherein the modified human STAT3 sensestrand is the oligonucleotide of claim 1, wherein the humanCpG(D19)-STAT3 antisense strand comprises (a) a first oligonucleotidehaving the nucleotides set forth in SEQ ID NO:16, (b) a C3 carbon chainor propanediol linker and (c) a second oligonucleotide having thenucleotides set forth in SEQ ID NO:5 that is the antisense strand, andwherein the sense strand and the antisense strand anneal to form adouble stranded siRNA.
 3. A chimeric molecule comprising a human STAT3sense strand-overhang, a human CpG(D19)-overhang and a human STAT3antisense strand, wherein the human STAT3 sense strand-overhangcomprises (a) an oligonucleotide having the nucleotides set forth in SEQID NO:18 that is the sense strand, (b) a C3 carbon chain or propanediollinker and (c) an oligonucleotide having the nucleotides set forth inSEQ ID NO:19 that is the overhang, wherein nucleotides at positions 2,4, 7, 8 and 11-15 of SEQ ID NO:19 are modified, wherein the humanCpG(D19)-overhang comprises (a) an oligonucleotide having thenucleotides set forth in SEQ ID NO:16, (b) a C3 carbon chain orpropanediol linker and (c) an oligonucleotide having the nucleotides setforth in SEQ ID NO:20 that is the overhang, wherein nucleotides atpositions 7, 8, 11, 12, 14 and 15 of SEQ ID NO:20 are modified, whereinthe human STAT3 antisense strand comprises an oligonucleotide having thenucleotides set forth in SEQ ID NO:5, wherein the modifications areindependently selected from the group consisting of a 2′-alkylpyrimidine, 2′F, 2′OMe, amino, a deoxynucleotide, an abasic sugar, a2-O-alkyl modified pyrimidine, 4-thiouracil, 5-bromouracil,5-iodouracil, 5-(3-aminoallyl)-uracil and LNA, wherein the overhangsanneal to form a double stranded RNA, and wherein the sense strand andthe antisense strand anneal to form a double stranded siRNA.